WO1997045559A1 - Detection de differences dans des sequences d'acides nucleiques utilisant une combinaison de la detection par ligase et de reactions d'amplification en chaine par polymerase - Google Patents

Detection de differences dans des sequences d'acides nucleiques utilisant une combinaison de la detection par ligase et de reactions d'amplification en chaine par polymerase Download PDF

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WO1997045559A1
WO1997045559A1 PCT/US1997/009012 US9709012W WO9745559A1 WO 1997045559 A1 WO1997045559 A1 WO 1997045559A1 US 9709012 W US9709012 W US 9709012W WO 9745559 A1 WO9745559 A1 WO 9745559A1
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oligonucleotide
primer
sequences
polymerase chain
chain reaction
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PCT/US1997/009012
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Phillip Belgrader
Francis Barany
Matthew Lubin
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Cornell Research Foundation, Inc.
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Priority to AU32160/97A priority Critical patent/AU730633B2/en
Priority to JP54287897A priority patent/JP4468488B2/ja
Priority to CA002255774A priority patent/CA2255774C/fr
Priority to EP97927787A priority patent/EP0912761A4/fr
Publication of WO1997045559A1 publication Critical patent/WO1997045559A1/fr

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Definitions

  • the present invention relates to the detection of nucleic acid sequence differences using coupled ligase detection reaction ("LDR”) and polymerase chain reaction (“PCR”).
  • LDR coupled ligase detection reaction
  • PCR polymerase chain reaction
  • One aspect of the present invention involves use of a ligase detection reaction coupled to a polymerase chain reaction.
  • Another aspect of the present invention relates to the use of a primary polymerase chain reaction coupled to a secondary polymerase chain reaction coupled to a ligase detection reaction.
  • a third aspect of the present invention involves a primary polymerase chain reaction coupled to a secondary polymerase chain reaction.
  • a variety of DNA hybridization techniques are available for detecting the presence of one or more selected polynucleotide sequences in a sample containing a large number of sequence regions.
  • a fragment containing a selected sequence is captured by hybridization to an immobilized probe.
  • the captured fragment can be labeled by hybridization to a second probe which contains a detectable reporter moiety.
  • Another widely used method is Southern blotting. In this method, a mixture of DNA fragments in a sample is fractionated by gel electrophoresis, then fixed on a nitrocellulose filter. By reacting the filter with one or more labeled probes under hybridization conditions, the presence of bands containing the probe sequences can be identified.
  • the method is especially useful for identifying fragments in a restriction-enzyme DNA digest which contains a given probe sequence and for analyzing restriction-fragment length polymorphisms ("RFLPs").
  • Another approach to detecting the presence of a given sequence or sequences in a polynucleotide sample involves selective amplification of the sequence(s) by polymerase chain reaction.
  • primers complementary to opposite end portions of the selected sequence(s) are used to promote, in conjunction with thermal cycling, successive rounds of primer-initiated replication.
  • the amplified sequence(s) may be readily identified by a variety of techniques. This approach is particularly useful for detecting the presence of low- copy sequences in a polynucleotide-containing sample, e.g. , for detecting pathogen sequences in a body-fluid sample.
  • the confronting ends of the probe elements can be joined by ligation, e.g. , by treatment with ligase.
  • the ligated probe element is then assayed, evidencing the presence of the target sequence.
  • the ligated probe elements act as a template for a pair of complementary probe elements.
  • the target sequence is amplified linearly, allowing very small amounts of target sequence to be detected and/or amplified.
  • This approach is referred to as ligase detection reaction.
  • the process is referred to as the ligase chain reaction which achieves exponential amplification of target sequences.
  • Jou, et. al. "Deletion Detection in Dystrophia Gene by Multiplex Gap Ligase Chain Reaction and Immunochromatographic Strip Technology, " Human Mutation 5:86-93 (1995) relates to the use of a so called “gap ligase chain reaction” process to amplify simultaneously selected regions of multiple exons with the amplified products being read on an immunochromatographic strip having antibodies specific to the different haptens on the probes for each exon.
  • Solid-phase hybridization assays require multiple liquid-handling steps, and some incubation and wash temperatures must be carefully controlled to keep the stringency needed for single-nucleotide mismatch discrimination. Multiplexing of this approach has proven difficult as optimal hybridization conditions vary greatly among probe sequences.
  • Allele-specific PCR products generally have the same size, and an assay result is scored by the presence or absence of the product band(s) in the gel lane associated with each reaction tube.
  • Gibbs et al. Nucleic Acids Res.. 17:2437-2448 (1989). This approach requires splitting the test sample among multiple reaction tubes with different primer combinations, multiplying assay cost.
  • PCR has also discriminated alleles by attaching different fluorescent dyes to competing allelic primers in a single reaction tube (F.F. Chehab, et al. , Proc. Natl. Acad. Sci. USA.
  • Ligation of allele-specific probes generally has used solid-phase capture (U. Landegren et al. , Science. 241 : 1077-1080 (1988); Nickerson et al. , Proc. Natl. Acad. Sci. USA. 87:8923-8927 (1990)) or size-dependent separation (D.Y. Wu, et al. , Genomics. 4:560-569 (1989) and F. Barany, Proc. Natl. Acad. Sci.. 88: 189-193 (1991)) to resolve the allelic signals, the latter method being limited in multiplex scale by the narrow size range of ligation probes.
  • the ligase detection reaction alone cannot make enough product to detect and quantify small amounts of target sequences.
  • the gap ligase chain reaction process requires an additional step - polymerase extension.
  • the use of probes with distinctive ratios of charge/translational frictional drag for a more complex multiplex will either require longer electrophoresis times or the use of an alternate form of detection.
  • Tandem repeat DNA sequences known as microsatellites represent a very common and highly polymorphic class of genetic elements within the human genome. These microsatellite markers containing small repeat sequences have been used for primary gene mapping and linkage analysis. Weber, J.L. et al. , Am. J. Hum. Genet. 44: 388-396 (1989); Weissenbach, J. et al. , Nature (London) 359: 794-800 (1992). PCR amplification of these repeats allows rapid assessment for loss of heterozygosity and can greatly simplify procedures for mapping tumor suppressor genes. Ruppert, J.M. , et al. , Cancer Res. 53: 5093-94 (1993); van der Riet, et al.
  • PCR is commonly used for microsatellite analysis in identifying both the appearance of new polymorphisms and the loss of heterozygosity in cancer detection.
  • L. Mao, et. al. "Microsatellite Alterations as Clonal Markers for the Detection of Human Cancer, " Proc. Nat'l Acad. Sci USA 91(21): 9871-75 (1994); L. Mao, et. al. , "Molecular Detection of Primary Bladder Cancer by Microsatellite Analysis, " Science 271 :659-62 (1996); D. Radford, et. al. ,
  • PCR has also been used for human identification, such as paternity testing, criminal investigations, and military personnel identification.
  • A. Syvanen et. al. "Identification of Individuals by Analysis of Biallelic DNA Markers, Using PCR and Solid-Phase Mini-Sequencing" Am. J. Hum. Genet. 52(1): 46-59 (1993) describes a mini-sequencing approach to human identification. The technique requires PCR amplification of individual markers with at most 4 PCR reactions being carried out at a time in a single PCR tube. Mini-sequencing is carried out to determine individual polymorphisms.
  • the present invention is directed to the detection of nucleic acid sequence differences using coupled LDR and PCR processes.
  • the present invention can be carried out in one of the following 3 embodiments: (1) LDR coupled to PCR; (2) primary PCR coupled to secondary PCR coupled to LDR; and (3) primary PCR coupled to secondary PCR.
  • Each of these embodiments have particular applicability in detecting certain characteristics.
  • each requires the use of coupled reactions for multiplex detection of nucleic acid sequence differences where ohgonucleotides from an early phase of each process contain sequences which may be used by ohgonucleotides from a later phase of the process.
  • One aspect of the present invention relates to a method for identifying two or more of a plurality of sequences differing by one or more single-base changes, insertions, deletions, or translocations in a plurality of target nucleotide sequences.
  • This method involves a first polymerase chain reaction phase, a second polymerase chain reaction phase, and a ligase detection reaction phase. This process involves analyzing a sample potentially containing one or more target nucleotide sequences with a plurality of sequence differences.
  • each group comprises one or more primary oligonucleotide primer sets with each set having a first nucleotide primer, having a target-specific portion and a 5' upstream secondary primer-specific portion, and a second oligonucleotide primer, having a target-specific portion and a 5' upstream secondary primer-specific portion.
  • the first oligonucleotide primers of each set in the same group contain the same 5 ' upstream secondary primer- specific portion and the second oligonucleotide primers of each set in the same group contain the same 5' upstream primer-specific portion.
  • the oligonucleotide primers in a particular set are suitable for hybridization on complementary strands of a corresponding target nucleotide sequence to permit formation of a polymerase chain reaction product. However, there is a mismatch which interferes with formation of such a polymerase chain reaction product when the primary oligonucleotide primers hybridize to any other nucleotide sequence in the sample.
  • the polymerase chain reaction products in a particular set may be distinguished from other polymerase chain reaction products in the same group or groups.
  • the primary oligonucleotide primers, the sample, and the polymerase are blended to form a primary polymerase chain reaction mixture.
  • the primary polymerase chain reaction mixture is subjected to two or more polymerase chain reaction cycles involving a denaturation treatment, a hybridization treatment, and an extension treatment, as substantially described above.
  • target-specific portions of the primary oligonucleotide primers hybridize to the target nucleotide sequences.
  • the extension treatment causes hybridized primary oligonucleotide primers to be extended to form primary extension products complementary to the target nucleotide sequence to which the primary oligonucleotide primers are hybridized.
  • the upstream secondary primer-specific portions of a primary oligonucleotide primer set are not present on the target DNA, their sequences are copied by the second and subsequent cycles of the primary polymerase chain reaction phase.
  • the primary extension products produced after the second cycle have the secondary primer-specific portions on their 5' ends and the complement of primer-specific portion on their 3' ends.
  • This phase involves providing one or a plurality of secondary oligonucleotide primer sets. Each set has a first secondary oligonucleotide primer containing the same sequence as the 5' upstream portion of the first primary oligonucleotide primer, and a second secondary oligonucleotide primer containing the same sequence as the 5 ' upstream portion of the second primary oligonucleotide primer from the same primary oligonucleotide primer set as the first primary oligonucleotide complementary to the first secondary primer.
  • a set of secondary oligonucleotide primers may be used to amplify all of the primary extension products in a given group.
  • the secondary oligonucleotide primers are blended with the primary extension products and the polymerase to form a secondary polymerase chain reaction mixture.
  • the secondary polymerase chain reaction mixture is subjected to one or more polymerase chain reaction cycles having a denaturation treatment, a hybridization treatment, and an extension treatment, as substantially set forth above.
  • the hybridization treatment the secondary oligonucleotide primers hybridize to the complementary sequences present on the primary extension products but not to the original target sequence.
  • the extension treatment causes the hybridized secondary oligonucleotide primers to be extended to form secondary extension products complementary to the primary extension products.
  • the last phase of this aspect of the present invention involves a ligase detection reaction process.
  • a plurality of oligonucleotide probe sets are provided where each set has a first oligonucleotide probe, having a secondary extension product-specific portion and a detectable reporter label, and a second oligonucleotide probe, having a secondary extension product-specific portion.
  • the oligonucleotide probes in a particular set are suitable for ligation together when hybridized adjacent to one another on a complementary secondary extension product-specific portion. However, there is a mismatch which interferes with such ligation when the oligonucleotide probes are hybridized to any other nucleotide sequence present in the sample.
  • the ligation product of oligonucleotide probes in a particular set may be distinguished from either probe or other ligation products.
  • the plurality of oligonucleotide probe sets, the secondary extension products, and a ligase are blended to form a ligase detection reaction mixture.
  • the ligase detection reaction mixture is subjected to one or more ligase detection reaction cycles having a denaturation treatment and hybridization treatment substantially as described above.
  • the oligonucleotide probe sets hybridize at adjacent positions in a base-specific manner to the respective secondary extension products if present. As a result, adjacent probes ligate to one another to form a ligation product sequence containing the detectable reporter label and the secondary extension product-specific portions connected together.
  • the oligonucleotide probe sets may hybridize to nucleotide sequences other than their respective complementary secondary extension products but do not ligate together due to the presence of one or more mismatches and individually separate during the denaturation treatment.
  • the primary PCR/secondary PCR/LDR process of the present invention provides significant advantages over the use of PCR alone in the multiplex detection of single nucleotide and tandem repeat polymorphisms.
  • PCR oligonucleotide primers
  • the quantity of oligonucleotide primers must be increased to detect greater numbers of target nucleotide sequences.
  • the probability of target independent reactions e.g. , the primer-dimer effect
  • the mutations must be known, false positives may be generated by polymerase extension off of normal template, closely-clustered sites due to interference of overlapping primers cannot undergo multiplex detection, single base or small insertions and deletions in small repeat sequences cannot be detected, and quantification of mutant DNA in high background of normal DNA is difficult.
  • the number of target nucleotide sequences detected in a single multiplex PCR process is limited.
  • Direct sequencing requires enrichment of mutant samples in order to correct sequences, requires multiple reactions for large genes containing many exons, requires electrophoretic separation of products, is time consuming, and cannot be used to detect mutant DNA in less than 5 % of background of normal DNA.
  • mini-sequencing the mutation must be known, closely-clustered sites due to interference of overlapping primers cannot undergo multiplex detection, single base or small insertions and deletions in small repeat sequences cannot be detected, and four separate reactions are required.
  • ASO allele-specific oligonucleotide hybridization
  • the mutation must be known, hybridization and washing conditions must be known, cross-reactivity is difficult to prevent, closely-clustered sites due to interference of overlapping primers cannot undergo multiplex detection, and mutant DNA cannot be detected in less than 5 % of background of normal DNA.
  • Primer-mediated RFLP requires electrophoretic separation to distinguish mutant from normal DNA, is of limited applicability to sites that may be converted into a restriction site, requires additional analysis to determine the nature of the mutation, and is difficult to use where the mutant DNA is in a high background of normal DNA.
  • SSCP Single strand conformational polymorphism analysis
  • ddF dideoxynucleotide finger printing
  • DGGE Denaturing gradient gel electrophoresis
  • RNase mismatch cleavage requires additional analysis to determine the nature of the mutation, requires analysis of both strands to exclude RNase-resistant mismatches, and imposes difficulty in detecting mutations in a high background of normal DNA.
  • Chemical mismatch cleavage cannot detect mutant DNA in less than 5% of background of normal DNA, and requires an analysis of both strands to detect all mutations.
  • the primary PCR phase produces primary extension products with a secondary primer-specific portion.
  • This initial phase is carried out under conditions effective to maximize production of primary extension products without obtaining the adverse effects sometimes achieved in a PCR-only process.
  • the primary PCR phase of the present invention is carried out with 15 to 20 PCR cycles and utilizes less primer than would be utilized in a PCR-only process.
  • the primary PCR phase of the present invention produces extension products in a varied and unpredictable way, because some target nucleotide sequences will be amplified well, while others will not.
  • the primary PCR/secondary PCR/LDR process of the present invention is able to achieve multiplex detection of hundreds of nucleotide sequence differences in a single tube without undue customization of operating conditions for each particular sample being analyzed. Since the selection of mutant sequences is mediated by LDR rather than PCR, the primary PCR/secondary PCR/LDR process is less susceptible to false-positive signal generation.
  • the primary PCR/secondary PCR/LDR process allows detection of closely-clustered mutations, detection of single base or small insertions and deletions in small repeat sequences, quantitative detection of less than 1 % mutations in high background of normal DNA, and detection of ligation product sequences using addressable arrays.
  • the only significant requirements are that the mutations be known and that a multitude of ohgonucleotides be synthesized.
  • the ability to detect single nucleotide and tandem repeat polymorphisms is particularly important for forensic DNA identification and diagnosis of genetic diseases.
  • a second aspect of the present invention relates to a method for identifying one or more of a plurality of sequences differing by one or more single-base changes, insertions, deletions, or translocations in a plurality of target nucleotide sequences.
  • This method has a ligase detection reaction phase followed by a polymerase chain reaction phase.
  • This method involves providing a sample potentially containing one or more target nucleotide sequences with a plurality of sequence differences.
  • the ligase detection reaction phase one or more oligonucleotide probe sets are provided.
  • Each set has a first oligonucleotide probe, having a target-specific portion and a 5' upstream primer-specific portion, and a second oligonucleotide probe, having a target-specific portion and a 3' downstream primer-specific portion.
  • the oligonucleotide probes in a particular set are suitable for ligation together when hybridized adjacent to one another on a corresponding target nucleotide sequence. However, there is a mismatch which interferes with such ligation when they are hybridized to any other nucleotide sequence present in the sample.
  • the sample, the plurality of oligonucleotide probe sets, and a ligase are blended together to form a ligase detection reaction mixture.
  • the ligase detection reaction mixture is subjected to one or more ligase detection reaction cycles. These cycles include a denaturation treatment and a hybridization treatment. In the denaturation treatment, any hybridized oligonucleotides are separated from the target nucleotide sequences.
  • the hybridization treatment causes the oligonucleotide probe sets to hybridize at adjacent positions in a base-specific manner to their respective target nucleotide sequences if present in the sample. Once hybridized, the oligonucleotide probe sets ligate to one another to form a ligation product sequence. This product contains the 5' upstream primer-specific portion, the target-specific portions connected together, and the 3' downstream primer-specific portion.
  • the ligation product sequence for each set is distinguishable from other nucleic acids in the ligase detection reaction mixture.
  • the oligonucleotide probe sets hybridized to nucleotide sequences in the sample other than their respective target nucleotide sequences but do not ligate together due to a presence of one or more mismatches and individually separate during the subsequent denaturation treatment.
  • one or a plurality of oligonucleotide primer sets are provided in the polymerase chain reaction. Each set has an upstream primer containing the same sequence as the 5 ' upstream primer-specific portion of the ligation product sequence and a downstream primer complementary to the 3 ' downstream primer-specific portion of the ligation product sequence, where one primer has a detectable reporter label.
  • the ligase detection reaction mixture is blended with the one or a plurality of oligonucleotide primer sets and the polymerase to form a polymerase chain reaction mixture.
  • the polymerase chain reaction mixture is subjected to one or more polymerase chain reaction cycles which include a denaturation treatment, a hybridization treatment, and an extension treatment.
  • a denaturation treatment hybridized nucleic acid sequences are separated.
  • the hybridization treatment causes primers to hybridize to their complementary primer-specific portions of the ligation product sequence.
  • hybridized primers are extended to form extension products complementary to the sequences to which the primers are hybridized.
  • the downstream primer hybridizes to the 3' downstream primer-specific portion of the ligation product sequence and is extended to form an extension product complementary to the ligation product sequence.
  • the upstream primer hybridizes to the 5' upstream primer-specific portion of the extension product complementary to the ligation product sequence and the downstream primer hybridizes to the 3 ' downstream portion of the ligation product sequence.
  • the reporter labels are detected and the extension products are distinguished to indicate the presence of one or more target nucleotide sequences in the sample.
  • One embodiment of the LDR/PCR process of the present invention achieves improved results over the use of LDR alone in measuring the number of gene copies in a cell (i.e. gene dosage).
  • LDR alone it is difficult to produce sufficient target copies which are needed ultimately to quantify a plurality of genes.
  • the LDR phase ligation product sequences are produced in a ratio proportional to the ratio of the genes from which they were derived within the sample.
  • the PCR phase amplifies ligation product sequences to the same degree so that their proportionality is maintained.
  • Target sequences originally found in the sample being analyzed are not amplified by the PCR phase, because such target sequences do not contain PCR primer- specific portions.
  • only the oligonucleotide primers for the PCR phase have reporter labels, only extension products with those labels will be detected.
  • Determination of variation in gene dosage is important in a number of biomedical applications.
  • the result (having one copy of an X-linked gene) is similar to the situation in male cells and is usually tolerated without manifestations of an inherited disorder. However, if the woman's son inherits her deleted X chromosome, he will have no copies of the genes in the deletion region and suffer from the X-linked disorder related to the absence of the gene.
  • the detection of chromosomal deletions therefore, is one application of the LDR/PCR process of the present invention.
  • Congenital chromosomal disorders occur when a fertilized egg has an abnormal compliment of chromosomes.
  • the most common congenital chromosomal disorder is Down Syndrome, which occurs when there is an additional chromosome 21 in each cell, designated 47,XX+21 or 47,XY+21.
  • the LDR/PCR process of the present invention can be designed to identify congenital chromosomal disorders.
  • the LDR/PCR process of the present invention is also useful for distinguishing polymorphisms in mono-nucleotide and di-nucleotide repeat sequences. It will also be useful in distinguishing minor populations of cells containing unique polymorphisms for detection of clonality.
  • the LDR/PCR process can be used with both gel and non-gel (i.e. DNA array) technologies. It allows multiplex analysis of many gene amplifications and deletions simultaneously, allows quantitative analysis, and does not require an external standard. The only relatively minor challenge presented by the LDR/PCR process is that it is difficult to use in determining the boundaries of large deletions in chromosomes.
  • microsatellite marker analysis cannot be used to detect small regions that are deleted or amplified, is not compatible with simultaneous detection of amplified regions, and depends on the availability of informative markers.
  • Competitive PCR i.e. differential PCR
  • Southern hybridization is time consuming, labor intensive, is not amenable to multiplexing due to the need for multiple steps for each probe tested, and requires large quantities of DNA.
  • Fluorescent in situ hybridization (“FISH”) requires specialized expertise, is time consuming, and requires large probes to analyze for suspected deleted or amplified regions.
  • FISH fluorescent in situ hybridization
  • a third aspect of the present invention also involves a method for identifying two or more of a plurality of sequences differing by one or more single-base changes, insertions, deletions, or translocations in one or more target nucleotide sequences. This method involves subjecting a sample potentially containing one or more target nucleotide sequences with a plurality of sequence differences to two successive polymerase chain reaction phases.
  • each group comprises two or more primary oligonucleotide primer sets.
  • Each set has a first oligonucleotide primer, having a target-specific portion and a 5' upstream secondary primary- specific portion, and a second oligonucleotide primer, having a target-specific portion and a 5' upstream secondary primer-specific portion.
  • the first oligonucleotide primers of each set in the same group contain the same 5' upstream secondary primer-specific portion and the second oligonucleotide primers of each set in the same group contain the same 5' upstream secondary primer- specific portion.
  • the oligonucleotide primers in a particular set are suitable for hybridization on complementary strands of a corresponding target nucleotide sequence to permit formation of a polymerase chain reaction product. However, there is a mismatch which interferes with formation of such a polymerase chain reaction product when the primary oligonucleotide primers hybridize to any other nucleotide sequence present in the sample.
  • the polymerase chain reaction products in a particular set may be distinguished from other polymerase chain reaction products with the same group or other groups.
  • the primary oligonucleotide primers are blended with the sample and the polymerase to form a primary polymerase chain reaction mixture.
  • the primary polymerase chain reaction mixture is subjected to two or more polymerase chain reaction cycles involving a denaturation treatment, a hybridization treatment, and an extension treatment, as described above.
  • a denaturation treatment a denaturation treatment
  • a hybridization treatment a hybridization treatment
  • an extension treatment a polymerase chain reaction cycle involving a denaturation treatment, a hybridization treatment, and an extension treatment, as described above.
  • the target-specific portion of a primary oligonucleotide primer is hybridized to the target nucleotide sequences.
  • the extension treatment the hybridized primary oligonucleotide primers are extended to form primary extension products complementary to the target nucleotide sequence to which the primary oligonucleotide primer is hybridized.
  • the upstream secondary primer-specific portions of a primary oligonucleotide primer set are not present on the target DNA, their sequences are copied by the second and subsequent cycles of the primary polymerase chain reaction phase.
  • the primary extension products produced after the second and subsequent cycles have the secondary primer- specific portions on their 5' ends and the complement of primer-specific portion on their 3' ends.
  • each set has a first secondary primer having a detectable reporter label and containing the same sequence as the 5' upstream portion of a first primary oligonucleotide primer, and a second secondary primer containing the same sequence as the 5' upstream primer of the second primary oligonucleotide primer from the same primary oligonucleotide primer set as the first primary oligonucleotide complementary to the first secondary primer.
  • a set of secondary oligonucleotide primers amplify the primary extension products in a given group.
  • the secondary oligonucleotide primers are blended with the primary extension products and the polymerase to form a secondary polymerase chain reaction mixture.
  • the secondary polymerase chain reaction mixture is subjected to one or more polymerase chain reaction cycles involving a denaturation treatment, a hybridization treatment, and an extension treatment, as described above.
  • the hybridization treatment the secondary oligonucleotide primers are hybridized to the primary extension products, while the extension treatment causes the hybridized secondary oligonucleotide primers to be extended to form secondary extension products complementary to the primary extension products.
  • the labelled secondary extension products are detected. This indicates the presence of one or more target nucleotide sequences in the sample.
  • the primary PCR/secondary PCR process of the present invention provides significant advantages over Southern hybridization, competitive PCR, and microsatellite marker analysis in detecting nucleotide deletions which cause a loss of heterozygosity.
  • Southern hybridization is more accurate than competitive PCR, it is quite labor intensive, requires large amounts of DNA, and neither technique can be multiplexed.
  • Current multiplex microsatellite marker approaches require careful attention to primer concentrations and amplification conditions.
  • the primary PCR/secondary PCR process of the present invention overcomes these difficulties encountered in the prior art.
  • the primary oligonucleotide primers flank dinucleotide or other repeat sequences and include a secondary primer-specific portion.
  • the primary PCR phase is carried out at low concentrations of these primers to allow several loci to undergo amplification at the same time.
  • the secondary PCR phase causes amplification to continue at the same rate with target-specific secondary primers being selected to space one set of microsatellite markers from the adjacent set.
  • the primary PCR/secondary PCR process can be used to carry out multiplex detection in a single PCR tube and with single gel lane analysis.
  • This aspect of the present invention is useful in carrying out a microsatellite marker analysis to identify nucleotide deletions in a gene.
  • Such multiplex detection can be carried out in a single reaction tube.
  • the primary PCR/secondary PCR process cannot distinguish amplifications from deletions, so, when making such distinctions, this process must be used in conjunction with the above-described LDR/PCR process or a differential PCR process.
  • Figure 1 is a flow diagram depicting a primary PCR/secondary PCR/LDR process for detection of germline mutations, such as point mutations, by electrophoresis or capture on an addressable array.
  • the term "zip- code” which appears in Figure 1 and other drawings refers to a sequence specific to a subsequently used primer or probe but not to either the target sequence or other genome sequences.
  • Figure 2 is a flow diagram depicting a primary PCR/secondary PCR/LDR process for detection of biallelic polymorphisms by electrophoresis or capture on an addressable array.
  • Figure 3 is a flow diagram depicting a primary PCR/secondary PCR/LDR process for detection of cancer-associated mutations by electrophoresis or capture on an addressable array.
  • Figure 4 is a schematic diagram depicting a primary PCR/secondary PCR/LDR process for detection of biallelic polymorphisms.
  • Figure 5 is a schematic diagram depicting a primary PCR/secondary PCR/LDR process for detection of allelic differences using LDR oligonucleotide probes which distinguish all possible bases at a given site.
  • Figure 6 is a schematic diagram depicting a primary PCR/secondary PCR/LDR process for detection of the presence of any possible base at two nearby sites using LDR oligonucleotide probes which distinguish all possible bases at a given site.
  • Figure 7 is a schematic diagram depicting a primary PCR/secondary PCR/LDR process for detection of cancer-associated mutations at adjacent alleles.
  • Figure 8 is a flow diagram depicting an LDR/PCR process with and without restriction endonuclease digestion using electrophoresis detection.
  • Figure 9 is a flow diagram depicting an LDR/PCR process using detection on an addressable array using gene-specific addresses.
  • Figure 10 is a schematic diagram depicting an LDR/PCR process for multiplex detection of gene amplifications and deletions.
  • Figure 11 is a schematic diagram depicting an allele specific problem for an LDR/PCR process.
  • Figure 12 is a schematic diagram depicting a solution for the allele specific problem for an LDR/PCR process which is shown in Figure 11.
  • Figure 13 is a flow diagram depicting an LDR/PCR process with an intermediate exonuclease digestion phase for detection of biallelic polymorphisms by electrophoresis or capture on an addressable array.
  • Figure 14 is a flow diagram depicting an LDR/PCR process with an intermediate exonuclease digestion phase for detection of cancer-associated mutations by electrophoresis or capture on an addressable array.
  • Figure 15 is a schematic diagram depicting an LDR/PCR process with an intermediate exonuclease digestion phase for detection of allele specific mutations and polymorphisms.
  • Figure 16 is a schematic diagram depicting an LDR/PCR process with an intermediate exonuclease digestion phase for detection of mononucleotide repeat polymorphisms.
  • Figure 17 is a schematic diagram depicting an LDR/PCR process with an intermediate exonuclease digestion phase for detection of mononucleotide repeat polymorphisms which are in low abundance.
  • Figure 18 is a flow diagram depicting the detection of polymorphisms using an LDR/PCR process with an intermediate sequenase extension phase and a uracil N-glycosylase digestion phase after the LDR phase and before the PCR phase and with detection by electrophoresis or an addressable array.
  • Figure 19 is a flow diagram depicting the detection of cancer using an LDR/PCR process with an intermediate sequenase extension phase and a uracil N-glycosylase digestion phase after the LDR phase and before the PCR phase and with detection by electrophoresis or an addressable array.
  • Figure 20 is a schematic diagram depicting detection of mononucleotide repeats using an LDR/PCR process with an intermediate sequenase amplification phase and a uracil N-glycosylase digestion phase after the LDR phase and before the PCR phase.
  • Figure 21 is a schematic diagram depicting detection of mononucleotide repeat polymorphisms which are in low abundance using an LDR/PCR process with an intermediate sequenase amplification phase and a uracil N-glycosylase digestion phase after the LDR phase and before the PCR phase.
  • Figure 22 is a flow diagram depicting a primary PCR/secondary
  • Figure 23 is a schematic diagram depicting a primary PCR/secondary PCR process for multiplex detection of insertions and deletions in microsatellite repeats.
  • Figure 24 shows the design of LDR oligonucleotide probes for quantification of gene amplifications and deletions in an LDR/PCR process.
  • Figures 25A-D show electropherogram results for an LDR/PCR process.
  • Figures 26A-C show electropherogram results for an LDR/PCR process of ErbB, G6PD, Int2, p53, and SOD gene segments from normal human female DNA and from DNA of the breast cancer cell line ZR-75-30 and the gastric carcinoma cell line SKGT-2.
  • the ErbB gene is known to be amplified in the cancer cell lines.
  • Target-specific ligation product sequences of 104 bp are generated in 10 cycles (94°C for 30 sec, 65°C for 4 min) of LDR using 500 femtomoles of each ligation primer, 50 ng of genomic DNA, 124 units of Thermus thermophillus ("Tth ") ligase, 2 ⁇ l of 10X buffer (0.2 M Tris, pH 8.5 and 0.1 M MgCl 2 ), 2 ⁇ l of 10 mM NAD, and 1 ⁇ l of 200 mM DTT in a volume of 20 ⁇ l.
  • Tth Thermus thermophillus
  • the ligation products are proportionally amplified in 26 cycles (94 C C for 15 sec, 60°C for 50 sec) of PCR by the addition of 30 ⁇ l of a solution containing 5 ⁇ l 10X Stoffel buffer (Perkin Elmer), 25 picomoles of each oligonucleotide primer, 2.5 units of Taq polymerase Stoffel fragment, and 8 ⁇ l of a solution 5 mM in each dNTP.
  • the products are digested with Haelll and HinPU to generate FAM-labeled products of 58 bp (ErbB (i.e.
  • the peak height and area for ErbB is consistently small in normal genomic DNA.
  • the peak height and area for ErbB are elevated when gene dosage is investigated in ZR-75- 30, a cell line with known ErbB amplification.
  • the gastric cell line, SKGT-2 shows dramatic amplification of the ErbB gene and a modest amplification of Int2.
  • the G6PD gene peak may be embedded in the large ErbB peak.
  • Figures 27A-C show electropherogram results for an LDR/PCR process to determine whether amplification of ErbB affected the relative peak heights of the other LDR oligonucleotide probes and PCR oligonucleotide primers for G6PD, Int2, p53, and SOD.
  • Figure 27A the gene dosage determination for the four loci in normal human female DNA is shown. Peak heights and areas for G6PD, Int2, p53, and SOD are similar, as they were in the experiment using all five LDR primers.
  • Figure 27B G6PD, Int2, and SOD analyzed in the ZR-75- 30 breast cancer cell line show similar relative peak heights, comparable to their appearance in normal female DNA.
  • Figures 28A-C show electropherogram results for the PCR phase of a primary PCR/secondary PCR/LDR process. Multiplex PCR amplification of 12 loci using primary PCR oligonucleotide primers produces approximately equal amounts of product. Over 80 gene regions with single-base polymorphisms were identified from the Human Genome Database. Twelve of these (see Table 10 and Figures 29A-H) were amplified in a primary PCR phase as follows: Long primary PCR oligonucleotide primers were designed to have gene-specific 3' ends and 5' ends complementary to one of two sets of secondary PCR oligonucleotide primers.
  • the upstream primary PCR oligonucleotide primers were synthesized with either FAM (i.e. 6-carboxyfluorescein; fluorescent dye used in sequencing and mutation detection) or TET (i.e. tetrachlorinated-6-carboxyfluorescein; fluorescent dye used in sequencing/mutation detection) fluorescent labels. All 24 base long primary PCR oligonucleotide primers were used at low concentration (2 picomole of each primer in 20 ⁇ l) in a 15 cycle primary PCR phase. After this, the two sets of secondary PCR oligonucleotide primers were added at higher concentrations (25 picomoles of each) and the secondary PCR phase was conducted for an additional 25 cycles. The products were separated on a 373 DNA Sequencer (Applied
  • Panel A shows the electropherogram results for the FAM- and TET- labelled products combined.
  • Panel B shows the FAM-labelled products alone.
  • Panel C shows the TET-labelled products alone. The process produces similar amounts of multiplexed products without the need to adjust carefully primer concentrations or PCR conditions.
  • Figures 29A-H show electropherogram results for the LDR phase of a primary PCR/secondary PCR/LDR process in detecting 12 biallelic genes for forensic identification.
  • the primary and secondary PCR phases for the 12 polymorphic genes were performed as described in Fig. 28A-C.
  • the secondary PCR oligonucleotide primers were not fluorescently labelled.
  • the secondary PCR process extension products were diluted into a ligase buffer containing 36 LDR oligonucleotide probes (one common and two discriminating primers for each locus).
  • LDR oligonucleotide probe sets were designed in two ways: (i) allele-specific oligonucleotide probes were of the same length but contained either the FAM or TET label; or (ii) the allele-specific oligonucleotide probes were both labelled with HEX (i.e. hexachlorinated-6-carboxyfluorescein; fluorescent dye used in sequencing and mutation detection) but differed in length by two basepairs. After 20 cycles of the LDR phase, the ligation product sequences were resolved using a 10% polyacrylamide sequencing gel on a 373 DNA Sequencer (Applied Biosystems). Panel A and E show the 12 loci PCR/LDR profiles of two individuals.
  • HEX i.e. hexachlorinated-6-carboxyfluorescein; fluorescent dye used in sequencing and mutation detection
  • Panels B, C, and D show, respectively, the FAM, TET, and HEX data for the individual in panel A.
  • Panels F, G, and H show, respectively, the FAM, TET, and HEX data for the individual in panel E.
  • the individual in panel A is homozygous only at locus 6 (ALDOB (i.e. aldolase B)) and locus 8 (IGF (i.e. insulin growth factor)).
  • the individual in panel E is heterozygous only at loci 3 (C6 (i.e. complement component C6)), 5 (NF1 (i.e. neurofibromatosis)), 6 (ALDOB), and 8 (IGF).
  • ADOB aldolase B
  • IGF insulin growth factor
  • One aspect of the present invention relates to a method for identifying two or more of a plurality of sequences differing by one or more single-base changes, insertions, deletions, or translocations in a plurality of target nucleotide sequences.
  • This method involves a first polymerase chain reaction phase, a second polymerase chain reaction phase, and a ligase detection reaction phase. This process involves analyzing a sample potentially containing one or more target nucleotide sequences with a plurality of sequence differences.
  • each group comprises one or more primary oligonucleotide primer sets with each set having a first oligonucleotide primer, having a target- specific portion and a 5' upstream secondary primer- specific portion, and a second oligonucleotide primer, having a target-specific portion and a 5' upstream secondary primer-specific portion.
  • the first oligonucleotide primers of each set in the same group contain the same 5' upstream secondary primer-specific portion and the second oligonucleotide primers of each set in the same group contain the same 5 ' upstream primer-specific portion.
  • the oligonucleotide primers in a particular set are suitable for hybridization on complementary strands of a corresponding target nucleotide sequence to permit formation of a polymerase chain reaction product. However, there is a mismatch which interferes with formation of such a polymerase chain reaction product when the primary oligonucleotide primers hybridize to any other nucleotide sequence present in the sample.
  • the polymerase chain reaction products in a particular set may be distinguished from other polymerase chain reaction products in the same group or groups.
  • the primary oligonucleotide primers, the sample, and the polymerase are blended to form a primary polymerase chain reaction mixture.
  • the primary polymerase chain reaction mixture is subjected to two or more polymerase chain reaction cycles involving a denaturation treatment, a hybridization treatment, and an extension treatment, as substantially described above.
  • target-specific portions of the primary oligonucleotide primers hybridize to the target nucleotide sequences.
  • the extension treatment causes hybridized primary oligonucleotide primers to be extended to form primary extension products complementary to the target nucleotide sequence to which the primary oligonucleotide primers are hybridized.
  • the upstream secondary primer-specific portions of a primary oligonucleotide primer set are not present on the target DNA, their sequences are copied by the second and subsequent cycles of the primary polymerase chain reaction phase.
  • the primary extension products produced after the second cycle have the secondary primer-specific portions on their 5' ends and the complement of the primer-specific portion on their 3' ends.
  • This phase involves providing one or a plurality of secondary oligonucleotide primer sets. Each set has a first secondary oligonucleotide primer containing the same sequence as the 5' upstream portion of the first primary oligonucleotide primer, and a second secondary oligonucleotide primer containing the same sequence as the 5 ' upstream portion of the second primary oligonucleotide primer from the same primary oligonucleotide primer set as the first primary oligonucleotide complementary to the first secondary primer.
  • a set of secondary oligonucleotide primers may be used to amplify all of the primary extension products in a given group.
  • the secondary oligonucleotide primers are blended with the primary extension products and the polymerase to form a secondary polymerase chain reaction mixture.
  • the secondary polymerase chain reaction mixture is subjected to one or more polymerase chain reaction cycles having a denaturation treatment, a hybridization treatment, and an extension treatment, as substantially set forth above.
  • the hybridization treatment the secondary oligonucleotide primers hybridize to complementary sequences present on the primary extension products but not to the original target sequence.
  • the extension treatment causes the hybridized secondary oligonucleotide primers to be extended to form secondary extension products complementary to the primary extension products.
  • the last phase of this aspect of the present invention involves a ligase detection reaction process.
  • a plurality of oligonucleotide probe sets are provided where each set has a first oligonucleotide probe, having a secondary extension product-specific portion and a detectable reporter label, and a second oligonucleotide probe, having a secondary extension product-specific portion.
  • the oligonucleotide probes in a particular set are suitable for ligation together when hybridized adjacent to one another on a complementary secondary extension product-specific portion. However, there is a mismatch which interferes with such ligation when the oligonucleotide probes are hybridized to any other nucleotide sequence present in the sample.
  • the ligation product of oligonucleotide probes in a particular set may be distinguished from either individual probes or other ligation products.
  • the plurality of oligonucleotide probe sets, the secondary extension products, and a ligase are blended to form a ligase detection reaction mixture.
  • the ligase detection reaction mixture is subjected to one or more ligase detection reaction cycles having a denaturation treatment and hybridization treatment substantially as described above.
  • the oligonucleotide probe sets hybridize at adjacent positions in a base-specific manner to the respective secondary extension products if present. As a result, adjacent probes ligate to one another to form a ligation product sequence containing the detectable reporter label and the secondary extension product-specific portions connected together.
  • the oligonucleotide probe sets may hybridize to nucleotide sequences other than the respective complementary secondary extension products but do not ligate together due a presence of one or more mismatches and individually separate during the denaturation treatment.
  • the reporter labels of the ligation product sequences are detected which indicates the presence of one or more target nucleotide sequences in the sample.
  • Figures 1 , 2, and 3 show flow diagrams of the primary PCR/secondary PCR/LDR process of the present invention utilizing either of two detection procedures.
  • One alternative involves use of capillary electrophoresis or gel electrophoresis and a fluorescent quantification procedure.
  • detection can be carried out by capture on an array of capture oligonucleotide addresses and fluorescent quantification.
  • Figure 1 relates to detection of germline mutations (e.g. , a point mutation), while Figure 2 detects biallelic polymorphisms, and Figure 3 shows the detection of cancer-associated mutations.
  • Figure 1 depicts the detection of a germline point mutation.
  • step 1 after DNA sample preparation, multiple exons are subjected to primary PCR amplification using Taq (i.e. Thermus aquaticus) polymerase under hot start conditions with oligonucleotide primers having a target-specific portion and a secondary primer-specific portion.
  • Taq polymerase may be inactivated by heating at 100° C for 10 min or by a freeze/thaw step.
  • the products of the primary PCR amplification phase are then subjected in step 2 to secondary PCR amplification using Taq polymerase under hot start conditions with the secondary oligonucleotide primers.
  • Taq polymerase may be inactivated by heating at 100° C for 10 min or by a freeze/thaw step.
  • products of the secondary PCR phase are then diluted 20-fold into fresh LDR buffer containing LDR oligonucleotide probes containing allele-specific portions and common portions.
  • Step 4 involves the LDR phase of the process which is initiated by addition of Taq ligase under hot start conditions. During LDR, oligonucleotide probes ligate to their adjacent oligonucleotide probes only in the presence of target sequence which gives perfect complementarity at the junction site.
  • the products may be detected in two different formats.
  • fluorescently-labeled LDR probes contain different length poly A or hexaethylene oxide tails.
  • each ligation product sequence resulting from ligation of two probes hybridized on normal DNA
  • each ligation product resulting from ligation of two probes hybridized on normal DNA
  • a germline mutation would generate a new peak on the electropherogram.
  • the LDR probes may be designed such that the germline mutation ligation product sequence migrates with the same mobility as a normal DNA ligation product sequence, but it is distinguished by a different fluorescent reporter. The size of the new peak will approximate the amount of the mutation present in the original sample; 0% for homozygous normal, 50% for heterozygous carrier, or 100% for homozygous mutant.
  • each allele-specific probe contains e.g. , 24 additional nucleotide bases on their 5' ends. These sequences are unique addressable sequences which will specifically hybridize to their complementary address sequences on an addressable array. In the LDR reaction, each allele- specific probe can ligate to its adjacent fluorescently labeled common probe in the presence of the corresponding target sequence.
  • step 1 after DNA sample preparation, multiple exons are subjected to primary PCR amplification using Taq polymerase under hot start conditions with oligonucleotide primers having a target-specific portion and a secondary primer-specific portion.
  • the products of the primary PCR amplification phase are then subjected in step 2 to secondary PCR amplification using Taq polymerase under hot start conditions with the secondary oligonucleotide primers.
  • Taq polymerase may be inactivated by heating at 100° C for 10 min or by a freeze/ thaw step.
  • products of the secondary PCR phase are then diluted 20-fold into fresh LDR buffer containing LDR oligonucleotide probes containing allele-specific portions and common portions.
  • Step 4 involves the LDR phase of the process which is initiated by addition of Taq ligase under hot start conditions.
  • oligonucleotide probes ligate to their adjacent oligonucleotide probes only in the presence of target sequence which gives perfect complementarity at the junction site.
  • fluorescently-labeled LDR probes contain different length poly A or hexaethylene oxide tails. Each ligation product sequence will have a slightly different length and mobility, such that several LDR products yield a ladder of peaks.
  • the LDR probes may be designed such that the ligation products for polymorphic alleles migrate at the same position but are distinguished by different fluorescent reporter groups. The size of the peaks will approximate the amount of each allele.
  • each oligonucleotide probe contains unique addressable sequences with e.g., 24 additional nucleotide bases on their 5' ends. These sequences will specifically hybridize to their complementary address sequences on an array of capture ohgonucleotides.
  • each allele-specific probe can ligate to its adjacent fluorescently labeled common probe in the presence of corresponding target sequence. Ligation product sequences corresponding to each allele are captured on the array, while unligated oligonucleotide probes are washed away.
  • the black dots indicate that both chromosomes have a given allele, the white dots show that neither chromosome has that allele, and the shaded dots indicate that one chromosome has a given allele.
  • Figure 3 depicts the detection of cancer-associated mutation.
  • step 1 after DNA sample preparation, multiple exons are subjected to primary PCR amplification using Taq polymerase under hot start conditions with oligonucleotide primers having a target-specific portion and a secondary primer-specific portion.
  • the products of the primary PCR amplification phase are then subjected in step 2 to secondary PCR amplification using Taq polymerase under hot start conditions with the secondary oligonucleotide primers.
  • Taq polymerase may be inactivated by heating at 100° C for 10 min or by a freeze/ thaw step. Fluorescent quantification of PCR products can be achieved using capillary or gel electrophoresis in step 3.
  • step 4 the products are spiked with a 1/100 dilution of marker DNA (for each of the fragments).
  • This DNA is homologous to wild type DNA, except it contains a mutation which is not observed in cancer cells, but which may be readily detected with the appropriate LDR probes.
  • step 5 the mixed DNA products in products of the secondary PCR phase are then diluted 20-fold into fresh LDR buffer containing LDR oligonucleotide probes containing allele-specific portions and common portions.
  • step 6 involves the LDR phase of the process which is initiated by addition of Taq ligase under hot start conditions. During LDR, oligonucleotide probes ligate to their adjacent oligonucleotide probes only in the presence of target sequence which gives perfect complementarity at the junction site.
  • the products may be detected in the same two formats discussed above.
  • step 7a products are separated by capillary or gel electrophoresis, and fluorescent signals are quantified. Ratios of mutant peaks to marker peaks give the approximate amount of cancer mutations present in the original sample divided by 100.
  • step 7b products are detected by specific hybridization to complementary sequences on an addressable array. Ratios of fluorescent signals in mutant dots to marker dots give the approximate amount of cancer mutations present in the original sample divided by 100.
  • two DNA fragments of interest are treated with the primary PCR/secondary PCR/LDR process of the present invention.
  • the double stranded DNA molecules are denatured to separate the strands. This is achieved by heating to a temperature of 80-105° C.
  • Low concentrations of primary PCR oligonucleotide primers, containing a 3' target- specific portion (shaded area) and 5' secondary primer-specific portion (black area) are then added and allowed to hybridize to the strands, typically at a temperature of 50-85° C.
  • a thermostable polymerase e.g.
  • Taq aquaticus polymerase is also added, and the temperature is then adjusted to 50-85° C to extend the primer along the length of the nucleic acid to which the primer is hybridized.
  • the resulting double stranded molecule is heated to a temperature of 80-105° C to denature the molecule and to separate the strands.
  • the products of the primary PCR phase are blended with secondary PCR oligonucleotide primers and allowed to hybridize to one another, typically at a temperature of 50-85° C.
  • the secondary oligonucleotide primers are usually used in higher concentrations than are the primary oligonucleotide primers.
  • Taq polymerase is also added, and the temperature is then adjusted to 50-85° C to extend the primer along the length of the primary PCR extension products to which the secondary oligonucleotide primer is hybridized.
  • the resulting double stranded molecule is heated to a temperature of 80-105° C to denature the molecule and to separate the strands.
  • the ligation detection reaction phase begins, as shown in Figure 4.
  • a ligase for example, as shown in Figure 4, a thermostable ligase like Thermus aquaticus ligase.
  • the oligonucleotide probes are then allowed to hybridize to the target nucleic acid molecule and ligate together, typically, at a temperature of 45-85°C, preferably, 65°C. When there is perfect complementarity at the ligation junction, the ohgonucleotides can be ligated together.
  • variable nucleotide is T or A
  • the presence of T in the target nucleotide sequence will cause the oligonucleotide probe with the FI reporter label to ligate to the common oligonucleotide probe with the 5' poly A tail A, cast and the presence of A in the target nucleotide sequence will cause the oligonucleotide probe with the F2 reporter label to ligate to the common oligonucleotide probe with A instruct.
  • variable nucleotide is A or G
  • the presence of T in the target nucleotide sequence will cause the oligonucleotide probe with F3AA reporter label (i.e.
  • the F3 reporter label coupled to 2 additional bases forming a 5' poly A spacer
  • the F3 reporter label coupled to 2 additional bases forming a 5' poly A spacer
  • the presence of C in the target nucleotide sequence will cause the oligonucleotide probe with the F3 reporter label to ligate to the common oligonucleotide probe with the 5' poly A tail A n+4
  • the material is again subjected to denaturation to separate the hybridized strands.
  • the hybridization/ligation and denaturation steps can be carried out through one or more cycles (e.g.
  • detection can be carried out on an addressable array instead of with gel or capillary electrophoresis.
  • addressable arrays require that the poly A tails on the LDR oligonucleotide probe not containing a reporter label (i.e. tails A n and A n+4 ) be replaced with different addressable array-specific oligonucleotide portions.
  • a solid support is provided with an array of capture ohgonucleotides, some of which are complementary to the different addressable array-specific oligonucleotide portions. Hybridization of these portions to their complementary capture oligonucleotide probes indicates the presence of a corresponding nucleotide difference.
  • FIG. 5 is a schematic diagram of a primary PCR/secondary PCR/LDR process, in accordance with the present invention, where any possible base in 2 DNA molecules of interest are distinguished.
  • the primary and secondary PCR processes are carried out in substantially the same way as described for Figure 4.
  • Appearance of fluorescent reporter labels FI , F2, F3, and F4 in conjunction with the left hand DNA molecule indicates the presence of the A, G, C, and T alleles in the DNA molecule, respectively.
  • equal amounts of the FI and F3 reporter labels indicates that the individual in question is heterozygous for the A and C alleles.
  • the same reporter label is used to indicate the presence of the different alleles; however, on each oligonucleotide probe with the distinguishing bases, there are different 5' poly A tails. More particularly, a 2 unit poly A tail, a 4 unit poly A tail, a 6 unit poly A tail, and an 8 unit poly A tail correspond to the T, C, G, and A alleles in the DNA molecule, respectively. As shown in Figure 5, equal amounts of the FI reporter label with the A 6 and A 4 tails indicates that the individual in question is heterozygous for the G and C alleles.
  • FIG. 6 is a schematic diagram of a primary PCR/secondary PCR/LDR process, in accordance with the present invention, for detecting the presence of any possible base at two nearby sites in DNA molecules of interest.
  • the primary and secondary PCR phases are carried out in substantially the same way as described for Figure 4.
  • the LDR probes are able to overlap, yet are still capable of ligating provided there is perfect complementarity at the junction. This distinguishes LDR from other approaches, such as allele-specific PCR where overlapping primers would interfere with one another.
  • the discriminating oligonucleotide probes contain the reporter label with the discriminating base on the 3' end of these probes.
  • the poly A tails are on the 3' end of common oligonucleotide probes.
  • the presence of equal amounts of ligation product sequences with reporter labels FI and F3 shows that the individual in question is heterozygous for the A and C alleles in the first position.
  • the presence of ligation product sequences with reporter labels F2, F3, and F4 shows that the individual in question is heterozygous for the G, C, and T alleles.
  • the presence of equal amounts of ligation product sequences with reporter label FI having the A 6 and A 4 tails indicates that at the first position, the individual in question is heterozygous for the G and C alleles.
  • the presence of the equal amounts of ligation product sequences with reporter label Fl having the A 8 and A 2 tails indicates that the individual in question is heterozygous for the A and T alleles.
  • Figure 7 is a schematic diagram depicting the use of the primary PCR/secondary PCR/LDR process of the present invention to detect a low abundance mutation in the presence of an excess of normal sequence.
  • DNA molecule is codon 12 of the K-ras gene, sequence GGT, which codes for glycine ("Gly").
  • GGT which codes for glycine
  • GAT which codes for aspartic acid
  • the LDR probes for wild-type (i.e. normal) sequences are missing from the reaction. If the normal LDR probes (with the discriminating base being G) were included, they would ligate to the common probes and overwhelm any signal coming from the mutant target.
  • Figure 7 shows codon 61 of the K-ras gene sequence CAG which codes for glutamine ("Gin”). A small percentage of the cells contain the C to G mutation in GAG, which codes for glutamic acid ("Glu"). Again, the LDR oligonucleotide probes do not include the C and A bases found in the wild type form to avoid overwhelming the mutant signal.
  • the existence of a ligation product sequence with fluorescent label F2 and the A n+4 tail indicates the presence of the glutamic acid encoding mutant.
  • a second aspect of the present invention relates to a method for identifying one or more of a plurality of sequences differing by one or more single-base changes, insertions, deletions, or translocations in a plurality of target nucleotide sequences.
  • This method has a ligase detection reaction phase followed by a polymerase chain reaction phase.
  • This method involves providing a sample potentially containing one or more target nucleotide sequences with a plurality of sequence differences.
  • oligonucleotide probe sets are provided. Each set has a first oligonucleotide probe, having a target-specific portion and a 5' upstream primer-specific portion, and a second oligonucleotide probe, having a target-specific portion and a 3' downstream primer-specific portion.
  • the oligonucleotide probes in a particular set are suitable for ligation together when hybridized adjacent to one another on a corresponding target nucleotide sequence. However, there is a mismatch which interferes with such ligation when they are hybridized to any other nucleotide sequence present in the sample.
  • the sample, the plurality of oligonucleotide probe sets, and a ligase are blended together to form a ligase detection reaction mixture.
  • the ligase detection reaction mixture is subjected to one or more ligase detection reaction cycles. These cycles include a denaturation treatment and a hybridization treatment. In the denaturation treatment, any hybridized ohgonucleotides are separated from the target nucleotide sequences.
  • the hybridization treatment causes the oligonucleotide probe sets to hybridize at adjacent positions in a base-specific manner to their respective target nucleotide sequences if present in the sample. Once hybridized, the oligonucleotide probe sets ligate to one another to form a ligation product sequence. This product contains the 5' upstream primer-specific portion, the target-specific portions connected together, and the 3' downstream primer-specific portion.
  • the ligation product sequence for each set is distinguishable from other nucleic acids in the ligase detection reaction mixture.
  • the oligonucleotide probe sets hybridized to nucleotide sequences in the sample other than their respective target nucleotide sequences but do not ligate together due to a presence of one or more mismatches and individually separate during the denaturation treatment.
  • oligonucleotide primer sets are provided. Each set has an upstream primer containing the same sequence as the 5' upstream primer-specific portion of the ligation product sequence and a downstream primer complementary to the 3' downstream primer- specific portion of the ligation product sequence, where one primer has a detectable reporter label.
  • the ligase detection reaction mixture is blended with the one or a plurality of oligonucleotide primer sets and the polymerase to form a polymerase chain reaction mixture.
  • the polymerase chain reaction mixture is subjected to one or more polymerase chain reaction cycles which include a denaturation treatment, a hybridization treatment, and an extension treatment.
  • a denaturation treatment hybridized nucleic acid sequences are separated.
  • the hybridization treatment causes primers to hybridize to their complementary primer-specific portions of the ligation product sequence.
  • hybridized primers are extended to form extension products complementary to the sequences to which the primers are hybridized.
  • the downstream primer hybridizes to the 3' downstream primer-specific portion of the ligation product sequence and is extended to form an extension product complementary to the ligation product sequence.
  • the upstream primer hybridizes to the 5' upstream primer-specific portion of the extension product complementary to the ligation product sequence and the 3' downstream primer hybridizes to the 3' downstream portion of the ligation product sequence.
  • FIG. 8 is a flow diagram depicting the LDR/PCR process of the present invention with or without restriction endonuclease digestion and using capillary electrophoresis detection.
  • a DNA sample is mixed with Taq ligase and oligonucleotide probes containing a target-specific portion and a primer- specific portion.
  • the mixture is subjected to an LDR process to produce ligation product sequences containing the ligated target-specific portions and the primer- specific portions.
  • Step 2 involves mixing the ligation product sequences with Taq polymerase and primers and subjecting the mixture to a PCR process.
  • the next step is determined as a function of whether the ligation product sequences are the same or different sizes.
  • step 3a is selected which involves subjecting the extension products from PCR to capillary electrophoresis or gel electrophoresis, either of which is followed by fluorescent quantification.
  • Step 3b is utilized where the ligation product sequences are the same size and involves subjecting the extension products from the PCR phase to restriction endonuclease digestion.
  • step 4b This generates digestion fragments of unique size which can be subjected to capillary electrophoresis or gel electrophoresis, followed by fluorescent quantification, according to step 4b.
  • the curve generated as a result of electrophoresis shows three ligation product sequences migrating at lengths of 104, 107, and 110, with the peak areas representing amplification of the Her-2 gene, loss of heterozygosity of the p53 gene, and the control SOD gene, respectively.
  • the electrophoresis curve where steps 3b and 4b are used involves three ligation product sequence restriction fragments at lengths of 58, 70, and 76, with the peak areas representing amplification of the Her-2 gene, loss of heterozygosity of the p53 gene, and the control SOD gene, respectively.
  • Figure 9 shows the LDR/PCR process of the present invention where, in step 3, the extension products are captured on an array of capture oligonucleotide addresses.
  • the capture oligonucleotide probes can be complementary to a nucleotide sequence across the ligation junction.
  • the number of gene copies captured on the array of capture ohgonucleotides is then determined by fluorescent quantification as compared with known controls.
  • such analysis of the array indicates ligation product sequences hybridizing to gene-specific addresses, where the fluorescent intensity represents amplification of the Her-2 gene, loss of heterozygosity of the p53 gene, and the control SOD gene, respectively.
  • Figure 10 is a schematic diagram depicting an LDR/PCR process for multiplex detection of gene amplifications and deletions.
  • the ratio of the Her-2/neu gene from Chromosome 17q, the p53 gene from Chromosome 17p, and the SOD gene from Chromosome 21q is detected.
  • pairs of oligonucleotide probes, having a target-specific portion and a primer-specific portion are allowed to anneal adjacent to each other on target nucleic acids and ligate to one another (in the absence of mismatches).
  • This ligase detection reaction is carried out with Tth ligase at a hybridization/ligation temperature of 65° C which is well below the T m values of 75° C for the oligonucleotide probes.
  • the ligation product sequences are simultaneously amplified by PCR using Taq polymerase and two common primers complementary to the primer-specific portion, one of which is fluorescently labeled. This maintains the proportionality of the target sequences initially present in the sample.
  • the extension products are then digested with H ⁇ elll and Hmpll which releases fluorescently labeled fragments of unique sizes for each target sequence present in the sample. The digestion products are separated and analyzed on an Applied Biosystems, Inc. (Foster City, Calif.) 373A DNA Sequencer. The peak heights and areas are related to the relative copies of genes present in the initial target sample.
  • Figure 11 is a schematic diagram, depicting a problem which can be encountered with the allele-specific LDR/PCR process. While a PCR/LDR process is very powerful, there may be circumstances where a multiplexed allele- specific LDR/PCR process of the present invention would be preferred.
  • the concept is to have one or more sets of LDR oligonucleotide probes, each set characterized by (a) a first oligonucleotide probe, having a target-specific portion and a 5' upstream primer-specific portion, and (b) a second oligonucleotide probe, having a target-specific portion and a 3' downstream primer-specific portion.
  • the LDR oligonucleotide probes anneal adjacent to each other on the target sequence.
  • An LDR reaction using thermostable ligase black dot would form a ligation product sequence provided there is perfect complementarity at the ligation junction.
  • the ligation product sequences are PCR amplified with primer sets, each set characterized by (a) an upstream primer containing the same sequence as the 5' upstream primer-specific portion of a ligation product sequence and (b) a downstream primer complementary to the 3' downstream primer-specific portion of that ligation product sequence.
  • the primers are shown as black lines in step 2.
  • one primer is fluorescently labeled, it will generate a fluorescent product which may be detected in a variety of detection schemes.
  • a PCR extension product should not be formed in the absence of a ligation event.
  • polymerase can make several complementary copies of the downstream LDR probe using the downstream primer.
  • this downstream LDR probe extension product can anneal to the upstream LDR probe extension product off the target sequence, and generate a sequence containing the target region flanked by the two primer-specific sequences. This product will amplify as the LDR product, and thus yield a false positive signal.
  • Figure 12 is a schematic drawing showing a solution to the allele specific LDR/PCR problem, utilizing an intermediate exonuclease digestion step. Allele-specific LDR/PCR can be achieved while significantly reducing background ligation independent (incorrect) target amplification. To do so, it is necessary to eliminate one or more of the components required for ligation independent PCR amplification, without removing the information content of the ligation product sequence.
  • One solution is to use exonuclease in step 2 to digest unreacted LDR oligonucleotide probes from step 1. By blocking the end which is not ligated, for example the 3' end of the downstream oligonucleotide probe, one probe can be made substantially resistant to digestion, while the other is sensitive.
  • Blocking groups include use of a thiophosphate group and/or use of 2-O-methyl ribose sugar groups in the backbone.
  • Exonucleases include Exo I (3'-5'), Exo III (3'-5'), and Exo IV (both 5'-3' and 3'-5'), the later requiring blocking on both sides.
  • One convenient way to block both probes is by using one long "padlock" probe (see M. Nilsson et. al. , "Padlock Probes: Circularizing Ohgonucleotides for Localized DNA Detection, " Science 265: 2085-88 (1994), which is hereby incorporated by reference), although this is by no means required.
  • exonucleases for example a combination of Exo I (single strand specific) and Exo III (double strand specific), is the ability to destroy both target and one LDR probe, while leaving the ligation product sequences substantially undigested.
  • an exonuclease treatment prior to PCR in accordance with steps 3 and 4, either one or both oligonucleotide probes in each set are substantially reduced, and thus hybridization of the remaining oligonucleotide probes to the original target DNA (which is also substantially reduced by exonuclease treatment) and formation of a ligation product sequence which is a suitable substrate for PCR amplification by the oligonucleotide primer set is substantially reduced. In other words, formation of ligation independent labeled extension products is substantially reduced or eliminated.
  • FIG. 13 is a flow diagram showing an allele-specific LDR/PCR process using exonuclease digestion with either size based- or DNA array based- detection.
  • the flow diagram shows the three reactions required for the multiplexed allele-specific LDR/PCR process.
  • sets of LDR oligonucleotide probes (wherein the downstream probes are blocked on their 3' ends) are ligated in the presence of the correct allele target using Taq DNA ligase. Unreacted upstream probes are digested with exonuclease in step 2, coincidentally, target is also digested.
  • primer sets are used to amplify the ligation product sequence by hybridizing to the primer specific portions of ligation product sequences.
  • the LDR oligonucleotide probes in a given particular set generate a unique length product, and thus may be distinguished from either oligonucleotide probes or other ligation products.
  • the products are separated by size or electrophoretic mobility. Labels on the PCR primers are detected, and the products are distinguished by size.
  • the LDR oligonucleotide probes in a particular set use may be distinguished from either oligonucleotide probes or other ligation product sequences by differences in the sequences of the PCR primers.
  • each set characterized by (a) an upstream primer containing the same sequence as the 5' upstream primer-specific portion of a ligation product sequence, and (b) a downstream primer complementary to the 3' downstream primer-specific portion of that ligation product sequence, wherein one primer has a detectable reporter label and the other primer contains an addressable nucleotide sequence linked to the 5' end of that primer such that the addressable nucleotide sequence remains single stranded after a PCR reaction, all the products can be distinguished.
  • the latter may be achieved by using a non-natural base within a PCR primer which polymerase cannot extend through, thus generating PCR products which have single stranded tails. See C. Newton, et. al. , "The Production of PCR Products with 5' Single-stranded Tails Using Primers that
  • FIG 14 is a flow diagram showing a quantitative allele-specific LDR/PCR process using exonuclease digestion in step 3 with either size based- or DNA array based-detection.
  • the flow diagram shows how one can quantify the amounts of different targets (especially low abundance cancer mutations) by adding marker sequence(s) (step 1) at the start of the LDR reaction (step 2).
  • the biochemical reactions i.e. PCR (step 4)
  • the relative amount of mutant product to marker product are quantified using capillary or gel electrophoresis (step 5a) or capture on an addressable array (step 5b).
  • the amount of mutant target present in the original sample can then be determined.
  • FIG 15 is a schematic drawing showing an allele-specific LDR/PCR process with exonuclease digestion (step 2) for detection of mutations or polymorphisms. Mutations and polymo ⁇ hisms may be distinguished as described in Figure 12.
  • the upstream LDR oligonucleotide probes which have the discriminating allele-specific base at the 3' end of the target- specific portion, have different 5' upstream primer-specific portions.
  • different primers in the PCR amplification step (i.e. step 3)
  • An array based detection scheme may also be used, where the upstream (allele-specific) probes have different 5' upstream primer-specific portions, and the different PCR primers contain different addressable nucleotide sequences which remain single stranded after a PCR reaction.
  • Figure 16 is a schematic drawing showing an allele-specific LDR (step 1)/PCR (step 3) process using exonuclease digestion (step 2) for detection of mononucleotide or dinucleotide repeat polymo ⁇ hisms.
  • LDR/PCR is for detecting nucleotide repeat polymo ⁇ hisms, a task which cannot be achieved by allele-specific PCR (because the 3' nucleotide is always the same), nor easily achieved by observing PCR product size variation (due to Taq polymerase slippage during amplification).
  • the LDR oligonucleotide probes distinguish between an A 9 and A, 0 mononucleotide repeat sequence by virtue of the specificity of thermostable DNA ligase. LDR products are only formed on the correct length target sequence, and thus the presence of that target is distinguished (step 4).
  • Figure 17 is schematic drawing showing an allele-specific
  • LDR/PCR process using exonuclease digestion (step 2) for detection of low abundance mononucleotide or dinucleotide repeat mutations.
  • Mononucleotide repeat length mutations may be distinguished as described in Figure 12.
  • the LDR oligonucleotide probes (step 1) distinguish between an A 8 , A 9 (mutants), and A 10 (normal) mononucleotide repeat sequences by virtue of the specificity of thermostable DNA ligase.
  • the two upstream LDR oligonucleotide probes differ in the length of the mononucleotide sequence at their 3 ' ends of their target specific portion and have different 5' upstream primer-specific portions.
  • different primers in the PCR amplification step (step 3) may be labeled with different fluorescent groups (Fam and Tet) to allow for distinction of products (step 4).
  • This has the distinct advantage of allowing mononucleotide repeat polymo ⁇ hisms to be distinguished based on fluorescent label instead of size, the latter being susceptible to false positives due to polymerase slippage.
  • An array based detection scheme may also be used, where the upstream (allele-specific) probes have different 5' upstream primer-specific portions, and the different PCR primers contain different addressable nucleotide sequences which remain single stranded after a PCR reaction.
  • Figure 18 is a flow diagram, showing an allele-specific LDR/PCR process using uracil N-glycosylase selection with either size based- or DNA array based-detection.
  • the flow diagram shows the four reactions required for multiplexed allele-specific LDR/PCR.
  • Sets of LDR oligonucleotide probes (wherein one or both probes contain deoxy-uracil in place of deoxythimidine) are ligated in the presence of the correct allele target using Taq DNA ligase in step 1.
  • a complementary copy of the ligation product sequence is made with sequenase in step 2.
  • Sequenase is a modified T7 polymerase, with any easily inactivated polymerase (i.e.
  • PCR primer sets are used to amplify the sequenase extension products in step 4.
  • the LDR oligonucleotide probes in a particular set generate a unique length product, and thus may be distinguished from either probes or other ligation products.
  • the products are separated by size or electrophoretic mobility. Labels on the PCR primers are detected, and products are distinguished by size.
  • the LDR oligonucleotide probes in a particular set may be distinguished from either probes or other ligation product sequences by differences in the sequences of the primer-specific portions.
  • each set characterized by (a) an upstream primer containing the same sequence as the 5' upstream primer-specific portion of a ligation product sequence, and (b) a downstream primer complementary to the 3' downstream primer-specific portion of that ligation product sequence.
  • One primer has a detectable reporter label, and the other primer contains an addressable array-specific portion linked to the 5' end of that primer such that the addressable array-specific portion remains single stranded after a PCR reaction, one can distinguish all the products.
  • FIG. 19 is a flow diagram showing a quantitative allele-specific LDR/PCR process using uracil N-glycosylase selection with either size based- or DNA array based-detection.
  • the flow diagram shows how one can quantify the amounts of different targets (especially low abundance cancer mutations) by adding marker sequence(s) in step 1 at the start of the LDR phase in step 2.
  • the biochemical reactions i.e. sequenase treatment (step 3), uracil N-glycosylase selection (step 4), and PCR (step 5)
  • step 3 the biochemical reactions
  • step 4 uracil N-glycosylase selection
  • step 5 PCR
  • the relative amount of mutant product to marker product is quantified using capillary or gel electrophoresis (step 6a) or capture on an addressable array (step 6b). From this information, the amount of mutant target present in the original sample can be determined.
  • FIG 20 is a schematic drawing showing an allele-specific LDR/PCR process using uracil N-glycosylase selection (step 3) (after sequenase treatment (step 2)) for detection of mononucleotide or dinucleotide repeat polymo ⁇ hisms.
  • step 3 after sequenase treatment (step 2)) for detection of mononucleotide or dinucleotide repeat polymo ⁇ hisms.
  • One of the most powerful uses of the LDR/PCR process is for detecting nucleotide repeat polymo ⁇ hisms, a task which cannot be achieved by allele-specific PCR (since the 3' nucleotide is always the same), nor easily achieved by observing PCR product size variation (due to Taq polymerase slippage during amplification), as in step 4.
  • the LDR (step 1) oligonucleotide probes distinguish between an A 9 and A 10 mononucleotide repeat sequence by virtue of the specificity of thermostable DNA ligase, Ligation product sequences are only formed on the correct length target sequence, and, thus, the presence of that target is distinguished in step 5.
  • Figure 21 is a schematic drawing showing an allele-specific LDR/PCR process using uracil N-glycosylase selection for detection of low abundance mononucleotide or dinucleotide repeat mutations.
  • Mononucleotide repeat length mutations may be distinguished as described in Figure 18.
  • the LDR oligonucleotide probes distinguish between an A 8 , A 9 (mutants), and A, 0 (normal) mononucleotide repeat sequences by virtue of the specificity of thermostable DNA ligase (step 1). Sequenase treatment (step 2) and uracil N-glycosylase selection (step 3) are then carried out.
  • the two upstream LDR oligonucleotide probes differ in the length of the mononucleotide sequence at the 3' ends of their target-specific portion, and have different 5' upstream primer- specific portions.
  • different primers in the PCR amplification step (steps 4- 5)
  • Fam and Tet fluorescent groups
  • This has the distinct advantage of allowing one to distinguish mononucleotide repeat polymo ⁇ hisms based on fluorescent label instead of size, the latter being susceptible to false positives due to polymerase slippage.
  • An array based detection scheme may also be used, where the upstream (allele-specific) probes have different 5' upstream primer-specific portions, and the different PCR primers contain different addressable array-specific portions which remain single stranded after a PCR reaction.
  • the LDR/exonuclease/PCR process described with reference to Figures 11 to 17 and the LDR/sequenase/uracil N-glycosylase/PCR process set forth in Figures 18-21 provide the ability to multiplex detect and then PCR amplify many different target sequences and to distinguish multiple single-base or sequence variations, all in a single reaction tube. This is achieved by combining the sensitivity of PCR with the selectivity of LDR. Since the selection of mutant sequences is mediated by LDR rather than PCR, the primary PCR/secondary PCR/LDR process is less susceptible to false-positive signal generation.
  • the primary PCR/secondary PCR/LDR process allows detection of closely-clustered mutations, detection of single base or small insertions and deletions in small repeat sequences, quantitative detection of less than 1 % mutations in high background of normal DNA, and detection of ligation product sequences using addressable arrays. Detection of single base or small insertions and deletions in small and medium repeat sequences may cause "stutter" when the primary amplification is PCR. No other currently-available technique can adequately solve this problem, especially when the target sequence containing the mononucleotide repeat polymo ⁇ hism is present in a lower abundance than normal DNA. In fact, analysis of genomic mutations which involve repeat sequence changes is severely hampered by the PCR "stutter" problem.
  • a third aspect of the present invention also involves a method for identifying two or more of a plurality of sequences differing by one or more single-base changes, insertions, deletions, or translocations in one or more target nucleotide sequences. This method involves subjecting a sample potentially containing one or more target nucleotide sequences with a plurality of sequence differences to two successive polymerase chain reaction phases.
  • each group comprises one or more primary oligonucleotide primer sets.
  • Each set has a first oligonucleotide primer, having a target-specific portion and a 5' upstream secondary primary- specific portion, and a second oligonucleotide primer, having a target-specific portion and a 5' upstream secondary primer-specific portion.
  • the first oligonucleotide primers of each set in the same group contain the same 5 ' upstream secondary primer- specific portion and the second oligonucleotide primers of each set in the same group contain the same 5' upstream secondary primer- specific portion.
  • the oligonucleotide primers in a particular set are suitable for hybridization on complementary strands of a corresponding target nucleotide sequence to permit formation of a polymerase chain reaction product. However, there is a mismatch which interferes with formation of such a polymerase chain reaction product when the primary oligonucleotide primers hybridize to any other nucleotide sequence present in the sample.
  • the polymerase chain reaction products in a particular set may be distinguished from other polymerase chain reaction products with the same group or other groups.
  • the primary oligonucleotide primers are blended with the sample and the polymerase to form a primary polymerase chain reaction mixture.
  • the primary polymerase chain reaction mixture is subjected to two or more polymerase chain reaction cycles involving a denaturation treatment, a hybridization treatment, and an extension treatment, as described above.
  • the target-specific portion of a primary oligonucleotide primer is hybridized to the target nucleotide sequences.
  • the extension treatment the hybridized primary oligonucleotide primers are extended to form primary extension products complementary to the target nucleotide sequence to which the primary oligonucleotide primer is hybridized.
  • the upstream secondary primer-specific portions of a primary oligonucleotide primer set are not present on the target DNA, their sequences are copied by the second and subsequent cycles of the primary polymerase chain reaction phase.
  • the primary extension products produced after the second cycle have the secondary primer-specific portions on their 5' ends and the complement of primer-specific portion on their 3' ends.
  • each set has a first secondary primer having a detectable reporter label and containing the same sequence as the 5' upstream portion of a first primary oligonucleotide primer, and a second secondary primer containing the same sequence as the 5' upstream primer of the second primary oligonucleotide primer from the same primary oligonucleotide primer set as the first primary oligonucleotide complementary to the first secondary primer.
  • a set of secondary oligonucleotide primers amplify the primary extension products in a given group.
  • the secondary oligonucleotide primers are blended with the primary extension products and the polymerase to form a secondary polymerase chain reaction mixture.
  • the secondary polymerase chain reaction mixture is subjected to one or more polymerase chain reaction cycles involving a denaturation treatment, a hybridization treatment, and an extension treatment, as described above.
  • the hybridization treatment the secondary oligonucleotide primers are hybridized to the primary extension products, while the extension treatment causes the hybridized secondary oligonucleotide primers to be extended to form secondary extension products complementary to the primary extension products.
  • the labelled secondary extension products are detected. This indicates the presence of one or more target nucleotide sequences in the sample.
  • Figure 22 is a flow diagram depicting a primary PCR/secondary
  • step 1 i.e. the primary PCR phase
  • step 2 involves a secondary PCR phase where Taq polymerase is used to amplify the primary PCR extension products with oligonucleotide primers containing the same sequence as the secondary primer- specific portion of the primary PCR primers.
  • the extension products resulting from the secondary PCR phase are subjected in step 3 to capillary electrophoresis or gel electrophoresis, followed by fluorescent quantification.
  • FIG. 22 is a schematic diagram depicting a primary PCR/secondary PCR process, according to the present invention, for detection of the loss of heterozygosity due to insertions and deletions in microsatellite repeats.
  • the primary PCR phase in step 1 is initiated by denaturing the sample DNA at 94° C.
  • Long PCR oligonucleotide primers having 3' ends complementary to unique DNA surrounding microsatellite repeat sequences and 5' ends containing the same sequence as one of two primers utilized in the secondary PCR phase, are then caused to anneal to target DNA at 65° C.
  • the primary PCR phase is carried out for 10-15 cycles.
  • the long primers utilized in the primary PCR phase can be multiplexed as long as they do not amplify alleles with overlapping length ranges. These reactions must be carried out on tumor and corresponding normal DNA to identify informative (i.e heterozygous) loci.
  • step 2 i.e secondary PCR amplification
  • primers complementary to the 5' ends of the primary PCR primers are then used to amplify the primary PCR extension products at nearly equal efficiency.
  • the secondary PCR extension products are then separated and analyzed by gel electrophoresis and an Applied Biosystems Inc. 373A DNA Sequencer using the Genescan 672 software package. Areas of loss of heterozygosity at informative loci are identified.
  • the analysis in Figure 23 shows the presence of both alleles (i.e. , chromosomes) containing RBI and NM23 and loss of heterozygosity (i.e. , loss of allele on one chromosome) for p53.
  • ligase detection reaction is described generally in WO 90/17239 to Barany et al. , F. Barany et al. , "Cloning, Overexpression and Nucleotide
  • the ligase detection reaction can use 2 sets of complementary ohgonucleotides. This is known as the ligase chain reaction which is described in the 3 immediately preceding references, which are hereby inco ⁇ orated by reference.
  • the ligase detection reaction can involve a single cycle which is known as the oligonucleotide ligation assay. See Landegren, et al. , "A Ligase-Mediated Gene Detection Technique, " Science 241 : 1077-80 (1988); Landegren, et al. , “DNA Diagnostics — Molecular Techniques and Automation, " Science 242:229-37 (1988); and U.S. Patent No. 4,988,617 to Landegren, et al. , which are hereby inco ⁇ orated by reference During ligase detection reaction phases, the denaturation treatment is carried out at a temperature of 80-105° C, while hybridization takes place at 50- 85° C.
  • Each cycle comprises a denaturation treatment and a thermal hybridization treatment which in total is from about one to five minutes long.
  • the ligation detection reaction involves repeatedly denaturing and hybridizing for 2 to 50 cycles.
  • the total time for the ligase detection reaction phase is 1 to 250 minutes.
  • the oligonucleotide probe sets or primers can be in the form of ribonucleotides, deoxynucleotides, modified ribonucleotides, modified deoxyribonucleotides, modified phosphate-sugar-backbone ohgonucleotides, nucleotide analogs, and mixtures thereof.
  • the ohgonucleotides of the oligonucleotide probe sets each have a hybridization or melting temperature (i.e. T m ) of 66-70° C. These ohgonucleotides are 20-28 nucleotides long.
  • the oligonucleotide probe sets or primers have a reporter label suitable for detection.
  • Useful labels include chromophores, fluorescent moieties, enzymes, antigens, heavy metals, magnetic probes, dyes, phosphorescent groups, radioactive materials, chemiluminescent moieties, and electrochemical detecting moieties.
  • the polymerase chain reaction process is fully described in H. Erlich, et. al. , "Recent Advances in the Polymerase Chain Reaction," Science 252: 1643-50 (1991); M. Innis, et. al. , PCR Protocols: A Guide to Methods and Applications, Academic Press: New York (1990); and R. Saiki, et. al. , "Primer- directed Enzymatic Amplification of DNA with a Thermostable DNA
  • a particularly important aspect of the present invention is its capability to quantify the amount of target nucleotide sequence in a sample. This can be achieved in a number of ways by establishing standards which can be internal (i.e. where the standard establishing material is amplified and detected with the sample) or external (i.e. where the standard establishing material is not amplified, and is detected with the sample).
  • the signal generated by the reporter label, resulting from capture of ligation product sequences produced from the sample being analyzed are detected.
  • the strength of this signal is compared to a calibration curve produced from signals generated by capture of ligation product sequences in samples with known amounts of target nucleotide sequence.
  • This techniques involves use of an external standard.
  • Another quantification method in accordance with the present invention, relates to an internal standard.
  • a known amount of one or more marker target nucleotide sequences is added to the sample.
  • a plurality of marker-specific oligonucleotide probe sets are added along with the ligase, the previously-discussed oligonucleotide probe sets, and the sample to a mixture.
  • the marker-specific oligonucleotide probe sets have (1) a first oligonucleotide probe with a target-specific portion complementary to the marker target nucleotide sequence, and (2) a second oligonucleotide probe with a target-specific portion complementary to the marker target nucleotide sequence and a detectable reporter label.
  • the oligonucleotide probes in a particular marker-specific oligonucleotide set are suitable for ligation together when hybridized adjacent to one another on a corresponding marker target nucleotide sequence. However, there is a mismatch which interferes with such ligation when hybridized to any other nucleotide sequence present in the sample or added marker sequences.
  • the presence of ligation product sequences is identified by detection of reporter labels.
  • the amount of target nucleotide sequences in the sample is then determined by comparing the amount of ligation product sequence generated from known amounts of marker target nucleotide sequences with the amount of other ligation product sequences.
  • Another quantification method involves, analysis of a sample containing two or more of a plurality of target nucleotide sequences with a plurality of sequence differences.
  • ligation product sequences corresponding to the target nucleotide sequences are detected and distinguished by any of the previously-discussed techniques.
  • the relative amounts of the target nucleotide sequences in the sample are then quantified by comparing the relative amounts of captured ligation product sequences generated. This provides a quantitative measure of the relative level of the target nucleotide sequences in the sample.
  • thermostable ligase is that derived from Thermus aquaticus. This enzyme can be isolated from that organism. M. Takahashi, et al. , "Thermophillic DNA Ligase," J. Biol. Chem. 259:10041-47 (1984), which is hereby inco ⁇ orated by reference. Alternatively, it can be prepared recombinantiy. Procedures for such isolation as well as the recombinant production of Thermus aquaticus ligase (as well as Thermus themophilus ligase) are disclosed in WO 90/17239 to Barany, et. al., and F.
  • ligases include E. coli ligase, T4 ligase, and Pyococcus ligase.
  • the ligation detection reaction mixture may include a carrier DNA, such as salmon sperm DNA.
  • the hybridization step in the ligase detection reaction which is preferably a thermal hybridization treatment discriminates between nucleotide sequences based on a distinguishing nucleotide at the ligation junctions.
  • the difference between the target nucleotide sequences can be, for example, a single nucleic acid base difference, a nucleic acid deletion, a nucleic acid insertion, or rearrangement. Such sequence differences involving more than one base can also be detected.
  • the oligonucleotide probe sets have substantially the same length so that they hybridize to target nucleotide sequences at substantially similar hybridization conditions.
  • the process of the present invention is able to detect infectious diseases, genetic diseases, and cancer. It is also useful in environmental monitoring, forensics, and food science.
  • infectious diseases can be detected by the process of the present invention. Typically, these are caused by bacterial, viral, parasite, and fungal infectious agents. The resistance of various infectious agents to drugs can also be determined using the present invention.
  • Bacterial infectious agents which can be detected by the present invention include Escherichia coli, Salmonella, Shigella, Klebsiella, Pseudomonas, Listeria monocytogenes, Mycobacierium tuberculosis, Mycobacterium avium- intracellulare, Yersinia, Francisella, Pasteurella, Brucella, Clostridia, Bordetella pertussis, Bacteroides, Staphylococcus aureus, Streptococcus pneumonia, B- Hemolytic strep.
  • Fungal infectious agents which can be detected by the present invention include Cryptococcus neoformans, Blastomyces dermatitidis, Histoplasma capsulatum, Coccidioides immitis, Paracoccidioides brasiliensis, Candida albicans, Aspergillus fumigautus, Phycomycetes (Rhizopus), Sporothrix schenckii, Chromomycosis, and Maduromycosis.
  • Viral infectious agents which can be detected by the present invention include human immunodeficiency virus, human T-cell lymphocytotrophic virus, hepatitis viruses (e.g. , Hepatitis B Virus and Hepatitis C Virus), Epstein- Barr Virus, cytomegalovirus, human papillomaviruses, orthomyxo viruses, paramyxo viruses, adenoviruses, corona viruses, rhabdo viruses, polio viruses, toga viruses, bunya viruses, arena viruses, rubella viruses, and reo viruses.
  • human immunodeficiency virus e.g. , Hepatitis B Virus and Hepatitis C Virus
  • Epstein- Barr Virus Epstein- Barr Virus
  • cytomegalovirus human papillomaviruses
  • orthomyxo viruses paramyxo viruses
  • corona viruses corona viruses
  • rhabdo viruses polio viruses
  • toga viruses bunya viruses
  • Parasitic agents which can be detected by the present invention include Plasmodium falciparum, Plasmodium malaria, Plasmodium vivax, Plasmodium ovale, Onchoverva volvulus, Leishmania, Trypanosoma spp. , Schistosoma spp. , Entamoeba histolytica, Cryptosporidum, Giardia spp. , Trichimonas spp. , Balatidium coli, Wuchereria bancrofti, Toxoplasma spp.
  • the present invention is also useful for detection of drug resistance by infectious agents.
  • vancomycin-resistant Enterococcus faecium methicillin-resistant Staphylococcus aureus, penicillin-resistant Streptococcus pneumoniae, multi-drug resistant Mycobacterium tuberculosis, and AZT-resistant human immunodeficiency virus can all be identified with the present invention.
  • Genetic diseases can also be detected by the process of the present invention. This can be carried out by prenatal or post-natal screening for chromosomal and genetic aberrations or for genetic diseases.
  • detectable genetic diseases include: 21 hydroxylase deficiency, cystic fibrosis, Fragile X Syndrome, Turner Syndrome, Duchenne Muscular Dystrophy, Down Syndrome or other trisomies, heart disease, single gene diseases, HLA typing, phenylketonuria, sickle cell anemia, Tay-Sachs Disease, thalassemia, Klinefelter Syndrome, Huntington Disease, autoimmune diseases, lipidosis, obesity defects, hemophilia, inborn errors of metabolism, and diabetes.
  • Cancers which can be detected by the process of the present invention generally involve oncogenes, tumor suppressor genes, or genes involved in DNA amplification, replication, recombination, or repair.
  • oncogenes include: BRCAl gene, p53 gene, APC gene, Her2/Neu amplification, Bcr/Abl , K-ras gene, and human papillomavirus Types 16 and 18.
  • Various aspects of the present invention can be used to identify amplifications, large deletions as well as point mutations and small deletions/insertions of the above genes in the following common human cancers: leukemia, colon cancer, breast cancer, lung cancer, prostate cancer, brain tumors, central nervous system tumors, bladder tumors, melanomas, liver cancer, osteosarcoma and other bone cancers, testicular and ovarian carcinomas, head and neck tumors, and cervical neoplasms.
  • the present invention can be used for detection, identification, and monitoring of pathogenic and indigenous microorganisms in natural and engineered ecosystems and microcosms such as in municipal waste water purification systems and water reservoirs or in polluted areas undergoing bioremediation. It is also possible to detect plasmids containing genes that can metabolize xenobiotics, to monitor specific target microorganisms in population dynamic studies, or either to detect, identify, or monitor genetically modified microorganisms in the environment and in industrial plants.
  • the present invention can also be used in a variety of forensic areas, including for human identification for military personnel and criminal investigation, paternity testing and family relation analysis, HLA compatibility typing, and screening blood, sperm, or transplantation organs for contamination.
  • the present invention has a wide variety of applications. For example, it can be used for identification and characterization of production organisms such as yeast for production of beer, wine, cheese, yogurt, bread, etc.
  • Another area of use is with regard to quality control and certification of products and processes (e.g. , livestock, pasteurization, and meat processing) for contaminants.
  • Other uses include the characterization of plants, bulbs, and seeds for breeding pu ⁇ oses, identification of the presence of plant-specific pathogens, and detection and identification of veterinary infections.
  • the oligonucleotide probes are suitable for ligation together at a ligation junction when hybridized adjacent to one another on a corresponding target nucleotide sequence due to perfect complementarity at the ligation junction.
  • the oligonucleotide probes in the set are hybridized to any other nucleotide sequence present in the sample, there is a mismatch at a base at the ligation junction which interferes with ligation.
  • the mismatch is at the base adjacent the 3' base at the ligation junction.
  • the mismatch can be at the bases adjacent to bases at the ligation junction.
  • detection and quantification can be carried out using capillary or gel electrophoresis or on a solid support with an array capture ohgonucleotides.
  • the mixture is contacted with the solid support at a temperature of 45-90° C and for a time period of up to 60 minutes.
  • Hybridizations may be accelerated by adding cations, volume exclusion or chaotropic agents.
  • the array is washed sequentially with a low stringency wash buffer and then a high stringency wash buffer.
  • oligonucleotide sets and the capture ohgonucleotides be configured so that the oligonucleotide sets hybridize to the target nucleotide sequences at a temperature less than that which the capture ohgonucleotides hybridize to the addressable array-specific portions. Unless the ohgonucleotides are designed in this fashion, false positive signals may result due to capture of adjacent unreacted ohgonucleotides from the same oligonucleotide set which are hybridized to the target.
  • the capture ohgonucleotides can be in the form of ribonucleotides, deoxy ribonucleotides, modified ribonucleotides, modified deoxyribonucleotides, peptide nucleotide analogues, modified peptide nucleotide analogues, modified phosphate-sugar backbone ohgonucleotides, nucleotide analogues, and mixtures thereof.
  • the detection phase of the process involves scanning and identifying if LDR or PCR products have been produced and correlating the presence of such products to a presence or absence of the target nucleotide sequence in the test sample.
  • Scanning can be carried out by scanning electron microscopy, confocal microscopy, charge-coupled device, scanning tunneling electron microscopy, infrared microscopy, atomic force microscopy, electrical conductance, and fluorescent or phosphor imaging. Correlating is carried out with a computer.
  • Genomic DNA was prepared from the blood of two normal human volunteers, one male and one female, according to standard techniques. Briefly, approximately 12 ml of blood was obtained in EDTA-containing blood collection tubes. Red blood cells were lysed by mixing the blood samples with 4 volumes of lysis buffer (10 mM Tris pH 8.0, 10 mM EDTA). After 10 min on ice with occasional agitation, the suspensions were centrifuged and the supernatants were decanted. The white blood cell pellets were resuspended in 20 ml of lysis buffer, and the above process was repeated. Each cell pellet was then suspended in 15 ml of digestion buffer (50 mM Tris pH 8.0, 5 mM EDTA, 100 mM NaCl, 1 %
  • the DNA precipitates were washed twice in 0.75 ml volumes of 70% EtOH, briefly centrifuging each time to allow removal of the supernatants. After removing the supernatants for the second time, the remaining EtOH was allowed to evaporate and the DNA was suspended in 0.5 ml of TE (lOmM Tri- HC1 pH 8.0 containing ImM EDTA) solution. A fifth dilution of each DNA solution was also prepared in TE. To determine the concentrations of the one fifth DNA solutions, 1 , 2, and 4 ⁇ l aliquots of each were loaded on a 1 % agarose gel with a known amount of Hindlll digested lambda DNA as a control.
  • the gel was run at 150 Volts for 2 hours with ethidium bromide in the electrophoresis buffer. After photographing the gel and comparing the intensities of the DNA bands, the one fifth dilutions were judged to have concentrations of approximately 100 ng/ml.
  • DNA solutions extracted from various tumor cell lines were the generous gifts of other laboratories. The concentrations of these solutions were checked in a similar fashion and solutions of 100 ng/ml in TE were prepared.
  • 25 ⁇ l of the 100 ng/ ⁇ l solutions was mixed with 5 ⁇ l of 10X medium salt buffer (0.5 M NaCl, 0.1 M MgCl,, 0.1 M Tris, pH 8.0), 20 ⁇ l of water-ME (i.e.
  • ohgonucleotides were synthesized on a 394A DNA Synthesizer (Applied Biosystems Division of Perkin-Elmer Co ⁇ . , Foster City, CA.). Ohgonucleotides labeled with 6-FAM were synthesized using the manufacturer's suggested modifications to the synthesis cycle (Applied Biosystems Inc. , 1994) and were subsequently deprotected at 55 ' C for 4 hr. LDR ohgonucleotides were purified by ethanol precipitation after overnight deprotection at 55 ° C. The primer-specific portions of the ohgonucleotides used for PCR amplification were purified by polyacrylamide gel electrophoresis on 10% acrylamide/7M urea gels.
  • Ohgonucleotides were visualized after electrophoresis by UV shadowing against a lightening screen and excised from the gel (Applied Biosystems Inc. , 1992). They were then eluted overnight at 64 ° C in TNE (i.e. Tris-sodium EDTA) buffer (100 mM Tris/HCl pH 8.0 containing 500 mM NaCl and 5 mM EDTA) and recovered from the eluate using Sep Pak cartridges (Millipore Co ⁇ , Milford, MA.) following the manufacture's instructions.
  • TNE i.e. Tris-sodium EDTA
  • Ohgonucleotides were resuspended in 100 ⁇ 1 TE (i.e. 10 mM Tri- HC1 pH 8.0 containing ImM EDTA). Typical concentrations of these original LDR probe solutions are about 1 ⁇ g/ ⁇ l or approximately 74 pm/ ⁇ l as determined by the following formula:
  • the concentrations of the LDR probes are given in Table 1.
  • the concentrations of ohgonucleotides complementary to the oligonucleotide probes of the ligase detection reaction were higher.
  • ZipALglF was 3.75 ⁇ g/ ⁇ l and ZipBLg2R was 2.01 ⁇ g/ ⁇ l or 524 pm/ ⁇ l and 281 pm/ ⁇ l, respectively, as determined by the formula above.
  • the downstream LDR ohgonucleotides probes were phosphorylated with T4 polynucleotide kinase. Aliquots of the 5 downstream ohgonucleotides equivalent to 200 pm (see Table 1) were combined with 10 ⁇ l of 10X kinase buffer (500 mM Tris/HCl pH 8.0, 100 mM MgCl 2 ), 10 ⁇ l of 10 mM ATP, 20 U T4 kinase, and sufficient water-ME to give a final volume of 100 ⁇ l. Phosphorylation was carried out at 37° C for 30 min followed by incubation for 10 min at 85 ° C to inactivate the T4 enzyme. The resulting concentration of the kinased LDR probe solution was 2 pm/ ⁇ l or 2000 fm/ ⁇ l in each probe.
  • 10X kinase buffer 500 mM Tris/HCl pH 8.0, 100 mM MgCl 2
  • 10 ⁇ l of 10 mM ATP
  • the kinase reaction is summarized as follows:
  • the solutions of the LDR and PCR ohgonucleotides were adjusted to convenient concentrations.
  • the kinased LDR probe solution was diluted fourfold in water to yield a concentration of 500fm/ ⁇ l.
  • a solution of the upstream LDR probes was made by combining volumes of the probes equivalent to 200 pm (see Table 1) with sufficient water to give a final volume of 400 ⁇ l. This created a solution 500 fm/ ⁇ l in each of the upstream LDR probes. Aliquots (20 ⁇ l) of the kinased and unkinased LDR probes were frozen for subsequent use.
  • Standard solutions of the PCR primers (10 pm/ ⁇ l) were prepared from their original solutions by combining 9.5 ⁇ l of ZipALglF and 17.8 ⁇ l of ZipBLg2R with sufficient water to achieve a total volume of 500 ⁇ l. These solutions were frozen for use in the LDR/PCR process. Unkinased probes were prepared according to the following:
  • Figure 24 shows the design of LDR oligonucleotide probes for quantification of gene amplifications and deletions in the LDR/PCR process.
  • These oligonucleotide probes were designed to recognize exon 8 in the p53 tumor suppressor gene (on chromosome 17p), exon 3 of int-2 (on chromosome l lq), an internal exon in HER-2/neu (i.e. HER-2/neu/erbB oncogene) (on chromosome 17q), exon 3 in SOD (i.e. super oxide dimutase) (on chromosome 21q), and exon 6 in G6PD (i.e.
  • Each pair of LDR oligonucleotide probes has the following features: (i) The left oligonucleotide probe contains from 5' to 3' an 18 base sequence identical to the fluorescently labeled secondary oligonucleotide primer (black bar), an "adjustment sequence" (white bar), and a target-specific sequence of from 22 to 28 bases with a T m of 75 °C (patterned bar); (ii) The right oligonucleotide probe contains from 5' to 3' a target-specific sequence of 20-25 bases with a T m of 75 °C (patterned bar), a single Haelll or HM > 1I restriction site at slightly different positions within the target-specific sequence, and an "adjustment sequence” (white bars).
  • the two oligonucleotide probes are designed such that their combined length is exactly 96 bases, with 50 G+C bases and 46 A+T bases.
  • the position of each unique restriction site generates a product which differs by at least 2 bases from the other products.
  • Each oligonucleotide probe set has an exon-specific region chosen to ligate the junction sequence of (A, T)C J C(A, T). This junction sequence corresponds to either a proline residue (codon CCN) or the complementary sequence of a tryptophan residue (TGG). These sequences were chosen to minimize differences in ligation rates and the chance of a polymo ⁇ hism at the ligation junction.
  • LDR Buffers/Reagents the following LDR buffers and reagents were selected:
  • 10X ST ligase buffer (0.2 M Tris pH 8.5, 0.1 M MgCl 2 ) [This was also tested with Tris at pH 7.6.]
  • 10 x TT ligase buffer (0.2 M Tris pH 7.6, 0.5 M KC1, 0.1 M MgCl 2 , 5mM EDTA) NAD (10 mM) DTT (200 mM) LDR primer solution containing one tenth concentration of each of the LDR primer mixtures (50 fm of each LDR primer per ⁇ l) Tth DNA Ligase (625 U/ ⁇ l)
  • PCR Buffers/Reagents the following PCR buffers and reagents were selected:
  • LDR was initiated by holding at 96 ° C for 2 minutes to denature the DNA followed by 10 cycles of 94 " C for 30 seconds and 65 " C for 4 minutes.
  • PCR reagent mixes for each reaction tube were constructed with the following proportions:
  • the G6PD peak was about half the area of other peaks, consistent with a single X-chromosome in males, while the other peaks were essentially the same.
  • the ErB2 peak for the NM10 Breast Cancer cell line is slightly elevated, while that in cell line SKBR3 is several fold greater than the normal female control, reflecting the known ErbB-2 gene amplification in these two cell lines.
  • cell line NM10 appears to have undergone LOH (i.e. a loss of heterozygosity) of p53
  • cell line SKBR3 appears to have undergone LOH of G6PD and p53. Some of the cells in cell line SKBR3 may have lost both copies of the p53 gene. Repeating these amplifications in the absence of the ErbB-2 primers was used to confirm the presence of these additional gene deletions (see below).
  • the ratios differ for each gene, (due to different efficiencies of LDR/PCR for each gene,) the ratios are generally consistent between the male and female sample, except for the G6PD/SOD ratio.
  • the G6PD for the female is about twice the value as the male, accurately reflecting the presence of two and one X chromosome, respectively.
  • the ErbB2 peak In the normal female, the ErbB2 peak is lower than the remaining 4 peaks. In different experiments, it was observed that the G6PD, Int-2, p53, and SOD peak areas would vary somewhat, but would retain the same relative profile from one sample to the next. See Figures 26A-C. The ErbB2 peak was consistently lower, and slight shoulders were observed on the G6PD and SOD peaks, for unknown reasons. The ErbB-2 peak in both cell line samples is several fold greater than the normal female control, reflecting the known ErbB-2 gene amplification in these two cell lines. In addition, the ZR-75-30 line appears to show LOH of p53, while the SKGT-2 cell line appears to have a slight amplification of the Int-2 region.
  • the ratios from using 4 sets of primers can be compared with 5 sets of primers to ascertain the internal consistency of this technique.
  • the values on the bottom half of Table 8 show the extent of ErbB-2 amplification. The numbers are quite consistent for the 4 primer and 5 primer amplifications (with the exception of SKGT2-G6PD noted above).
  • the ZR7530 cell line demonstrates a clear LOH for p53, while the SKGT2 cell line shows amplification of the Int-2 region, and both p53 genes present.
  • Ohgonucleotides were assembled by standard phosphoramidite chemistry on an Expedite DNA synthesizer (Perseptive Biosystems, Framingham, MA). Ohgonucleotides 5'-end labeled with 6-FAM, TET, and HEX were synthesized using the appropriate dye phosphoramidites (Perkin Elmer-Applied Biosystems) and purified with Oligonucleotide Purification Cartridges (Perkin Elmer- Applied Biosystems) following the manufacturer's protocol (Applied Biosystems Division-Perkin Elmer Co ⁇ .
  • Ohgonucleotides were resuspended in 250 ml TE (10 mM Tris/HCl and 5 mM EDTA pH 8.0). Typical concentrations were 300-500 mM for crude stock solutions and 100-200 mM for OPC (i.e. Oligonucleotide Purification Columns available from Applied Biosystems) purified stock solutions. For PCR and LDR, ohgonucleotides were diluted to working solutions of 10 mM (10 pmoles/ml) or 5 mM (5 pmoles/ml). Example 8 - Phosphorylation of LDR Ohgonucleotides
  • the 12 LDR common ohgonucleotides were phosphorylated at the 5' end to permit ligation to the fluorescent labeled ohgonucleotides.
  • the ohgonucleotides are shown below in Table 9.
  • the allele-specific ohgonucleotides are 5' end labeled with either FAM, TET, or HEX. All the common ohgonucleotides are phosphorylated at the 5' end Underline denotes tails that are not complementary to the target sequence.
  • LDR primer sets were designed in two ways, (i) allele-specific primers were of the same length hut contained either FAM or TET label; or ( ⁇ ) the allele-specific primers were both labeled with HEX but differed in length by two bases This was accomplished either during the synthesis with Phosphate- ON (Clontech Laboratories, Palo Alto, CA) according to the manufacturer's instructions or post-synthesis, using T4 polynucleotide kinase (Boehringer Mannheim, Indianapolis, IN) .
  • a common oligomer was diluted into 50 ⁇ l of kinase buffer (50 mM Tris/HCl, 10 mM MgCl 2 , and 1 mM ATP) to a final concentration of 1 mM (500 pmol in 50 ⁇ l ).
  • kinase buffer 50 mM Tris/HCl, 10 mM MgCl 2 , and 1 mM ATP
  • 1 mM 500 pmol in 50 ⁇ l
  • Ten units of T4 kinase was added, and the reaction was incubated at 37°C for 30 min. The T4 kinase was inactivated by heating at 95 ⁇ C for 10 min.
  • the kinase reaction was carried out as follows:
  • the site numbers are specific single point variations located within the respective genes All variations indicated are defined on the sense strand Genbank accession numbers are indicated in parentheses. Chr , chromosome; Het , heterozygosity
  • PCR amplifications were performed using genomic DNA isolated from whole blood using the Purgene DNA Isolation Kit (Gentra Systems, Inc. , Minneapolis, MI) according to the manufacturer's instructions.
  • PCR buffer 10 mM Tris/HCl pH 8.3, 10 mM KCI, 4 mM MgCl 2 , 0.4 mM each dNTP
  • 10-100 ng of genomic target DNA PCR hybrid primer pairs 1-12 (2 pmol of each primer)
  • PCR hybrid primer pairs 1-12 (2 pmol of each primer)
  • AmpliTaq DNA polymerase Stoffel fragment was placed in a thin- walled MicroAmp reaction tube (Applied Biosystems).
  • Forward and reverse hybrid primers for loci 1 ,3,5,7,10, and 12 contained 5' end regions identical to universal primers ALgl and BLg2 respectively.
  • Forward and reverse primers to loci 2,4,6,8,9, and 11 contained 5' end regions identical to universal primers CLg3 and DLg4, respectively.
  • the rationale for using a low concentration of the hybrid primers in the PCR phase was to deplete the hybrid primers during the reaction. This would theoretically allow products with low amplification efficiencies to "catch up with" those that had high amplification efficiencies. Amplification was attained by thermal cycling for 1 cycle of 96"C for 15 sec to denature, then 15 cycles of 94°C for 15 sec to denature and 65°C for 60 sec to anneal and extend.
  • the primary PCR process was carried out under the following conditions:
  • the primary PCR cycling conditions were as follows:
  • the secondary PCR process was carried out under the following conditions: 13.37 ⁇ l H 2 O
  • the secondary PCR cycling conditions were as follows: 94 °C 15" , 55 °C 1 ' x 25
  • the PCR products were separated on an Applied Biosystems 373 DNA sequencer.
  • a 3 ml aliquot of PCR sample was mixed with 3 ml of formamide containing fluorescently labeled Genescan-2500 [TAMRA] size standard (Applied Biosystems).
  • the formamide/standard solution was prepared by adding 50 ml Genescan-2500 size standard [TAMRAJ to 450 ml of formamide.
  • the sample was heated at 95°C for 2 min, quick cooled in ice, and electrophoresed through a denaturing 8% polyacrylamide gel in an Applied Biosystems 373 DNA sequencer running Genescan version 1.2 software.
  • the sizes of the fluorescently labeled products were automatically computed by the Genescan analysis software using the local Southern method.
  • the electropherograms clearly showed 12 distinct products (Figure 28).
  • the uniform amount of each product was attributed to the similar size of each amplicon and the use of the primary primers with portions complementary to the secondary primers, which annealed with identical affinities to the 12 amplicons without the need to carefully adjust reaction conditions.
  • the computed sizes of the products which ranged from 135 to 175 bp, matched exactly to their actual sizes (see Table 12).
  • PCR product amplified using unlabeled PCR universal primers served as the target for the LDR.
  • the polymerase in the PCR was inactivated by either freeze-thawing or adding EDTA/proteinase K to a final concentration of 5 mM and 100 mg/ml, respectively, and heating to 37°C for 30 min and 95°C for 10 min.
  • Proteinase K digestion was carried out under the following conditions:
  • PCR product Four microliters of PCR product was diluted in 20 ⁇ l of LDR mix containing 50 mM Tris/HCl pH 8.5, 50 mM KCl, 10 mM MgCl 2, 1 mM NAD + , 10 mM DTT, LDR oligonucleotide sets 1-12 (200 fmol of each oligonucleotide), and 10 units of Thermus aquaticus DNA ligase (Barany, F. and Gelfand, D. , "Cloning, Overexpression, and Nucleotide Sequence of a Thermostable DNA Ligase-Encoding Gene, " Gene. 109: 1-11 (1991), which is hereby incorporated by reference).
  • Each LDR oligonucleotide probe set consisted of two allele-specific ohgonucleotides and a common oligonucleotide.
  • Each pair of discriminating allele-specific oligonucleotide probes in the LDR oligonucleotide probe sets 1 ,2,3,4,6,7,8, and 9 were the same size, with one oligonucleotide labeled with 6- FAM and the other labeled with TET.
  • each pair of allele-specific ohgonucleotides differed by 2 bases (the larger oligonucleotide had a 5' tail that was not complementary to the target sequence), and both ohgonucleotides were labeled with HEX.
  • Thermal cycling was performed for 1 cycle of 95°C for 2 min to denature, then 20 cycles of 95°C for 30 sec to denature, and 65°C for 4 min to ligate.
  • the LDR process was carried out as follows:
  • the first individual ( Figure 29A-D; Table 13, individual 1) was heterozygous at polymorphic sites 1-7, and 10-12.
  • Heterozygosity at sites 1-4 and 6-7 was indicated by the detection of both 6-FAM and TET labeled products (Figure 29, panels B and C) at the respective positions on the electropherograms.
  • Heterozygosity at sites 5 and 11-12 was indicated by the presence of two HEX labeled products, differing in size by 2 bases, for each of these loci ( Figure 29, panel D).
  • the one product detected at sites 8 and 9 ( Figure 29, panels B and C) established that each of these loci was homozygous.
  • the second individual (Figure 29E-F and G-H; Table 13, individual 2) was heterozygous only at sites 3,5,6, and 9 and homozygous at sites 1 ,2,4,7,8, 10-12. There was a total of 8 differences in the genotypes at these positions between the two persons. Three additional individuals were typed, and all 5 persons had distinct genotypes based on the 12 loci (Table 13).

Abstract

Détection de différences dans des séquences d'acides nucléiques à l'aide d'une combinaison d'une réaction de détection par ligase et d'une réaction d'amplification en chaîne par polymérase. Selon un de ses aspects, la présente invention concerne une réaction de détection par ligase couplée à une réaction d'amplification en chaîne par polymérase. Selon un autre aspect, elle concerne l'utilisation d'une réaction primaire d'amplification en chaîne par polymérase couplée à une réaction secondaire d'amplification en chaîne par polymérase couplée à une réaction de détection par ligase. Selon un troisième aspect, elle concerne une réaction primaire d'amplification en chaîne par polymérase couplée à une réaction secondaire d'amplification en chaîne par polymérase. Ce couplage de la réaction de détection par ligase avec la réaction d'amplification en chaîne par polymérase permet une détection multiplex de différences dans des séquences d'acides nucléiques.
PCT/US1997/009012 1996-05-29 1997-05-27 Detection de differences dans des sequences d'acides nucleiques utilisant une combinaison de la detection par ligase et de reactions d'amplification en chaine par polymerase WO1997045559A1 (fr)

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CA002255774A CA2255774C (fr) 1996-05-29 1997-05-27 Detection de differences dans des sequences d'acides nucleiques utilisant une combinaison de la detection par ligase et de reactions d'amplification en chaine par polymerase
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Cited By (76)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0956359A1 (fr) * 1996-07-19 1999-11-17 Cornell Research Foundation, Inc. Detection tres precise de differences d'acides nucleiques par reaction de detection par ligase
WO2000055368A2 (fr) * 1999-03-15 2000-09-21 Pe Corporation (Ny) Complexes sonde/modificateur de mobilite pour detection d'acide nucleique multiplex
WO2001057268A2 (fr) * 2000-02-07 2001-08-09 Illumina, Inc. Detection d'acides nucleiques et procedes utilisant l'amorçage universel
WO2001057269A2 (fr) * 2000-02-07 2001-08-09 Illumina, Inc. Procedes de detection d'acide nucleique par amorçage universel
EP1130113A1 (fr) * 2000-02-15 2001-09-05 Johannes Petrus Schouten Méthode d'amplification dépendant de ligatures multiples
EP1136568A1 (fr) * 1999-10-04 2001-09-26 Olympus Optical Corporation Limited Procede de detection d'acide nucleique
WO2001085987A1 (fr) * 2000-05-09 2001-11-15 Diatech Pty. Ltd. Methodes d'identification des zones repetitives de polynucleotidiques d'une longueur determinee
WO2001092579A2 (fr) * 2000-05-30 2001-12-06 Pe Corporation (Ny) Methodes de detection d'acides nucleiques cibles au moyen d'une ligation et d'une amplification couplees
WO2002002815A1 (fr) * 2000-07-05 2002-01-10 Toyo Boseki Kabushiki Kaisha Procede de detection de polymorphismes nucleotidiques
EP1194592A1 (fr) * 1999-07-14 2002-04-10 Packard Bioscience Company Acides nucleiques modifies et utilisations associees
EP1221491A1 (fr) * 1999-10-12 2002-07-10 Precision System Science Co., Ltd. Systeme de suspension pour le sequen age d'une substance genetique, procede de sequen age d'une substance genetique mettant en oeuvre ledit systeme de suspension et procede d'identification des polymorphismes nucleotidiques uniques (snp) au moyen dudit systeme de suspension
EP1229128A1 (fr) * 2001-01-31 2002-08-07 Boehringer Mannheim Gmbh Nouveau procédé pour la détermination du génotype
US6458530B1 (en) 1996-04-04 2002-10-01 Affymetrix Inc. Selecting tag nucleic acids
EP1247815A2 (fr) * 2001-03-25 2002-10-09 Exiqon A/S Oligonucléotides modifiés et leurs utilisations
JP2003510011A (ja) * 1999-03-19 2003-03-18 コーネル リサーチ ファンデーション インク． カップル性ポリメラーゼ連鎖反応−制限エンドヌクレアーゼ消化−リガーゼ検出反応法
EP1319718A1 (fr) * 2001-12-14 2003-06-18 Keygene N.V. Analyse et détection de séquences cibles multiples à haut rendement
WO2003060163A2 (fr) * 2001-12-28 2003-07-24 Keygene N.V. Discrimination et detection de sequences nucleotidiques cibles utilisant la spectrometrie de masse
US6607878B2 (en) 1997-10-06 2003-08-19 Stratagene Collections of uniquely tagged molecules
EP1456416A2 (fr) * 2001-11-19 2004-09-15 Parallele Bioscience, Inc. Amplification multiplex cible par attache d'oligonucleotides
WO2005021794A2 (fr) * 2003-09-02 2005-03-10 Keygene N.V. Procedes bases sur l'amplification ou le dosage d'une ligation d'oligonucleotide (ola) permettant de detecter des sequences d'acide nucleique cibles
US6949370B1 (en) 1998-10-30 2005-09-27 Cornell Research Foundation, Inc. High fidelity thermostable ligase and uses thereof
WO2005092038A2 (fr) * 2004-03-22 2005-10-06 The Johns Hopkins University Procedes de detection de differences d'acides nucleiques
US6964847B1 (en) 1999-07-14 2005-11-15 Packard Biosciences Company Derivative nucleic acids and uses thereof
WO2005118862A2 (fr) * 2004-04-30 2005-12-15 Applera Corporation Compositions, techniques et kits permettant d'effectuer ou de mal effectuer une ligature d'oligoleotides
WO2006005055A2 (fr) * 2004-06-30 2006-01-12 Applera Corporation Procedes, melanges reactionnels et trousses destinees a la ligature de polynucleotides
EP1668148A1 (fr) * 2003-09-04 2006-06-14 Human Genetic Signatures PTY Ltd. Dosage de detection d'acide nucleique
EP1718766A2 (fr) * 2004-02-10 2006-11-08 Cornell Research Foundation, Inc. Procede de detection d'etat de methylation de promoteur
US7285384B2 (en) 2000-02-16 2007-10-23 Illuminia, Inc. Parallel genotyping of multiple patient samples
US7361488B2 (en) 2000-02-07 2008-04-22 Illumina, Inc. Nucleic acid detection methods using universal priming
WO2008118998A2 (fr) * 2007-03-27 2008-10-02 Primera Biosystems Inc. Procédé de détection multiplex et de quantification d'acides nucléiques
US7455965B2 (en) 2000-04-14 2008-11-25 Cornell Research Foundation, Inc. Method of designing addressable array for detection of nucleic acid sequence differences using ligase detection reaction
US7482443B2 (en) * 2000-03-09 2009-01-27 Genetag Technology, Inc. Systems and methods to quantify and amplify both signaling probes for cDNA chips and genes expression microarrays
EP2210665A1 (fr) 2002-01-16 2010-07-28 CLONDIAG GmbH Appareil de détection des molecules ciblées
US7799525B2 (en) 2003-06-17 2010-09-21 Human Genetic Signatures Pty Ltd. Methods for genome amplification
US7803580B2 (en) 2004-09-10 2010-09-28 Human Genetic Signatures Pty. Ltd. Amplification blocker comprising intercalating nucleic acids (INA) containing intercalating pseudonucleotides (IPN)
EP2241639A2 (fr) 2004-01-26 2010-10-20 CLONDIAG GmbH Procédé de génotypage et de pathotyage de Pseudomonas aeruginosa
US7833942B2 (en) 2004-12-03 2010-11-16 Human Genetic Signatures Pty. Ltd. Methods for simplifying microbial nucleic acids by chemical modification of cytosines
EP2256478A2 (fr) 2004-05-06 2010-12-01 CLONDIAG GmbH Dispositif et procede pour de detecter des interactions moleculaires
EP2266699A1 (fr) 2003-04-02 2010-12-29 CLONDIAG GmbH Appareil pour l'amplification et la détection d'acide nucléique
US7887752B2 (en) 2003-01-21 2011-02-15 Illumina, Inc. Chemical reaction monitor
EP2330215A1 (fr) 2005-11-04 2011-06-08 CLONDIAG GmbH Procédé et dispositif de détection d'interactions moléculaires
EP2356245A1 (fr) * 2008-11-07 2011-08-17 University Of Utah Research Foundation Biais d'amplification d'allèleallele amplification bias
US8168777B2 (en) 2004-04-29 2012-05-01 Human Genetic Signatures Pty. Ltd. Bisulphite reagent treatment of nucleic acid
US8236498B2 (en) 2006-01-20 2012-08-07 Olympus Corporation Method of detecting nucleotide sequence with an intramolecular probe
US8343738B2 (en) 2005-09-14 2013-01-01 Human Genetic Signatures Pty. Ltd. Assay for screening for potential cervical cancer
WO2013009175A1 (fr) 2011-07-08 2013-01-17 Keygene N.V. Génotypage à base de séquence en fonction d'analyses de ligature d'oligonucléotides
US8431347B2 (en) 2005-05-26 2013-04-30 Human Genetic Signatures Pty Ltd Isothermal strand displacement amplification using primers containing a non-regular base
WO2013095119A1 (fr) 2011-12-14 2013-06-27 De Staat Der Nederlanden, Vert. Door De Minister Van Vws Identification de souches de poliovirus
WO2013128281A1 (fr) * 2012-02-28 2013-09-06 Population Genetics Technologies Ltd Procédé de fixation d'une contre-séquence à un échantillon d'acides nucléiques
CN103388023A (zh) * 2012-05-11 2013-11-13 科美仪器 实时聚合酶链反应的集成分析系统和dna芯片以及使用其的集成分析方法
US8685675B2 (en) 2007-11-27 2014-04-01 Human Genetic Signatures Pty. Ltd. Enzymes for amplification and copying bisulphite modified nucleic acids
US8691754B2 (en) 2003-09-22 2014-04-08 Bioarray Solutions, Ltd. Microparticles with enhanced covalent binding capacity and their uses
US8691594B2 (en) 1996-04-25 2014-04-08 Bioarray Solutions, Ltd. Method of making a microbead array with attached biomolecules
US8906626B2 (en) 2000-02-07 2014-12-09 Illumina, Inc. Multiplex nucleic acid reactions
US8993271B2 (en) 1997-04-01 2015-03-31 Illumina, Inc. Method of nucleic acid amplification
EP2893034A1 (fr) * 2012-09-10 2015-07-15 Genesky Diagnostics (Suzhou) Inc. Procédé d'analyse d'acide nucléique multiplexe
US9147037B2 (en) 2004-08-02 2015-09-29 Bioarray Solutions, Ltd. Automated analysis of multiplexed probe-target interaction patterns: pattern matching and allele identification
WO2015185564A1 (fr) * 2014-06-02 2015-12-10 Base4 Innovation Ltd Procédé de détection de polymorphismes nucléotidiques
US9251583B2 (en) 2002-11-15 2016-02-02 Bioarray Solutions, Ltd. Analysis, secure access to, and transmission of array images
US9279148B2 (en) 1999-04-20 2016-03-08 Illumina, Inc. Detection of nucleic acid reactions on bead arrays
US9399795B2 (en) 1998-06-24 2016-07-26 Illumina, Inc. Multiplex decoding of array sensors with microspheres
WO2016121907A1 (fr) * 2015-01-30 2016-08-04 倉敷紡績株式会社 Procédé de préparation d'un produit d'adn monocaténaire
US9436088B2 (en) 2001-06-21 2016-09-06 Bioarray Solutions, Ltd. Un-supported polymeric film with embedded microbeads
US9637777B2 (en) 2003-10-28 2017-05-02 Bioarray Solutions, Ltd. Optimization of gene expression analysis using immobilized capture probes
US9709559B2 (en) 2000-06-21 2017-07-18 Bioarray Solutions, Ltd. Multianalyte molecular analysis using application-specific random particle arrays
CN107002145A (zh) * 2014-10-08 2017-08-01 康奈尔大学 用于使用针对遗留预防的组合核酸酶、连接反应和聚合酶反应鉴定和相对定量核酸序列表达、剪接变体、易位、拷贝数或甲基化变化的方法
US9732375B2 (en) 2011-09-07 2017-08-15 Human Genetic Signatures Pty. Ltd. Molecular detection assay using direct treatment with a bisulphite reagent
WO2018127408A1 (fr) 2017-01-05 2018-07-12 Tervisetehnoloogiate Arenduskeskus As Quantification de séquences d'adn
EP3378950A1 (fr) * 2017-03-21 2018-09-26 Sequencing Multiplex SLK Amplification et marquage simples en une étape
US10407717B2 (en) 2001-11-19 2019-09-10 Affymetrix, Inc. Methods of analysis of methylation
US10415081B2 (en) 2001-10-15 2019-09-17 Bioarray Solutions Ltd. Multiplexed analysis of polymorphic loci by concurrent interrogation and enzyme-mediated detection
WO2019238765A1 (fr) 2018-06-12 2019-12-19 Keygene N.V. Procédé d'amplification d'acide nucléique
WO2020169830A1 (fr) 2019-02-21 2020-08-27 Keygene N.V. Génotypage de polyploïdes
US11434502B2 (en) 2017-10-16 2022-09-06 Voyager Therapeutics, Inc. Treatment of amyotrophic lateral sclerosis (ALS)
US11542506B2 (en) 2014-11-14 2023-01-03 Voyager Therapeutics, Inc. Compositions and methods of treating amyotrophic lateral sclerosis (ALS)
US11603542B2 (en) 2017-05-05 2023-03-14 Voyager Therapeutics, Inc. Compositions and methods of treating amyotrophic lateral sclerosis (ALS)

Families Citing this family (674)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6652808B1 (en) * 1991-11-07 2003-11-25 Nanotronics, Inc. Methods for the electronic assembly and fabrication of devices
EP1382386A3 (fr) * 1992-02-19 2004-12-01 The Public Health Research Institute Of The City Of New York, Inc. Nouvelles configurations d'oligonucléotides et utilisation de ces configurations pour le tri, l'isolement, le sequençage et la manipulation des acides nucléiques
WO2000033244A2 (fr) * 1998-11-27 2000-06-08 Synaptics (Uk) Limited Capteur de position
US5710000A (en) * 1994-09-16 1998-01-20 Affymetrix, Inc. Capturing sequences adjacent to Type-IIs restriction sites for genomic library mapping
USRE43097E1 (en) 1994-10-13 2012-01-10 Illumina, Inc. Massively parallel signature sequencing by ligation of encoded adaptors
US6852487B1 (en) * 1996-02-09 2005-02-08 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using the ligase detection reaction with addressable arrays
US20020150921A1 (en) * 1996-02-09 2002-10-17 Francis Barany Detection of nucleic acid sequence differences using the ligase detection reaction with addressable arrays
US6881571B1 (en) * 1998-03-11 2005-04-19 Exonhit Therapeutics S.A. Qualitative differential screening
EP0912761A4 (fr) 1996-05-29 2004-06-09 Cornell Res Foundation Inc Detection de differences dans des sequences d'acides nucleiques utilisant une combinaison de la detection par ligase et de reactions d'amplification en chaine par polymerase
AU4220597A (en) * 1996-09-13 1998-04-02 Laboratory Of Molecular Biophotonics Solid phase for target nucleic acid detection, process for production thereof, and method of target nucleic acid detection
GB9624165D0 (en) * 1996-11-19 1997-01-08 Amdex A S Use of nucleic acids bound to carrier macromolecules
CA2276462C (fr) 1996-12-31 2007-06-12 Genometrix Incorporated Procede et dispositif d'analyse moleculaire multiplexee
US6327410B1 (en) 1997-03-14 2001-12-04 The Trustees Of Tufts College Target analyte sensors utilizing Microspheres
US20030027126A1 (en) 1997-03-14 2003-02-06 Walt David R. Methods for detecting target analytes and enzymatic reactions
US7622294B2 (en) * 1997-03-14 2009-11-24 Trustees Of Tufts College Methods for detecting target analytes and enzymatic reactions
US6143496A (en) 1997-04-17 2000-11-07 Cytonix Corporation Method of sampling, amplifying and quantifying segment of nucleic acid, polymerase chain reaction assembly having nanoliter-sized sample chambers, and method of filling assembly
EP0985142A4 (fr) 1997-05-23 2006-09-13 Lynx Therapeutics Inc Systeme et appareil destines au traitement sequentiel des analytes
US7348181B2 (en) 1997-10-06 2008-03-25 Trustees Of Tufts College Self-encoding sensor with microspheres
US7115884B1 (en) 1997-10-06 2006-10-03 Trustees Of Tufts College Self-encoding fiber optic sensor
GB9725197D0 (en) * 1997-11-29 1998-01-28 Secr Defence Detection system
US20030096232A1 (en) * 1997-12-19 2003-05-22 Kris Richard M. High throughput assay system
US20030039967A1 (en) * 1997-12-19 2003-02-27 Kris Richard M. High throughput assay system using mass spectrometry
US20100105572A1 (en) * 1997-12-19 2010-04-29 Kris Richard M High throughput assay system
US20020177144A1 (en) * 1997-12-30 2002-11-28 Jose Remacle Detection and/or quantification method of a target molecule by a binding with a capture molecule fixed on the surface of a disc
US20050053962A1 (en) * 1998-01-27 2005-03-10 Gary Blackburn Amplification of nucleic acids with electronic detection
US6759192B1 (en) * 1998-06-05 2004-07-06 Genset S.A. Polymorphic markers of prostate carcinoma tumor antigen-1(PCTA-1)
US20040203078A1 (en) * 1998-07-22 2004-10-14 National Institute Of Advanced Industrial Science And Technology Labeled complex, process for producing same and process for utilizing same
US6703228B1 (en) 1998-09-25 2004-03-09 Massachusetts Institute Of Technology Methods and products related to genotyping and DNA analysis
EP1001037A3 (fr) * 1998-09-28 2003-10-01 Whitehead Institute For Biomedical Research Pre-selection et isolement d'un polymorphisme de mononucleotide
US7014994B1 (en) 1999-03-19 2006-03-21 Cornell Research Foundation,Inc. Coupled polymerase chain reaction-restriction-endonuclease digestion-ligase detection reaction process
US6506594B1 (en) * 1999-03-19 2003-01-14 Cornell Res Foundation Inc Detection of nucleic acid sequence differences using the ligase detection reaction with addressable arrays
US20030215821A1 (en) * 1999-04-20 2003-11-20 Kevin Gunderson Detection of nucleic acid reactions on bead arrays
US6355431B1 (en) 1999-04-20 2002-03-12 Illumina, Inc. Detection of nucleic acid amplification reactions using bead arrays
US20030096321A1 (en) * 1999-05-19 2003-05-22 Jose Remacle Method for the identification and/or the quantification of a target compound obtained from a biological sample upon chips
US7056661B2 (en) * 1999-05-19 2006-06-06 Cornell Research Foundation, Inc. Method for sequencing nucleic acid molecules
US6544732B1 (en) * 1999-05-20 2003-04-08 Illumina, Inc. Encoding and decoding of array sensors utilizing nanocrystals
EP1190100B1 (fr) 1999-05-20 2012-07-25 Illumina, Inc. Decodage combinatoire de jeux d'acides nucleiques aleatoires
US8080380B2 (en) * 1999-05-21 2011-12-20 Illumina, Inc. Use of microfluidic systems in the detection of target analytes using microsphere arrays
US8481268B2 (en) 1999-05-21 2013-07-09 Illumina, Inc. Use of microfluidic systems in the detection of target analytes using microsphere arrays
JP3911909B2 (ja) * 1999-06-09 2007-05-09 株式会社日立製作所 Ｄｎａ試料調製方法及びｄｎａ試料調製装置
US20020119448A1 (en) 1999-06-23 2002-08-29 Joseph A. Sorge Methods of enriching for and identifying polymorphisms
US6692915B1 (en) 1999-07-22 2004-02-17 Girish N. Nallur Sequencing a polynucleotide on a generic chip
DK1218545T3 (da) 1999-08-18 2012-02-20 Illumina Inc Fremgangsmåder til fremstilling af oligonukleotidopløsninger
KR100858465B1 (ko) * 1999-09-10 2008-09-16 제론 코포레이션 올리고뉴클레오티드 엔3'→피5' 티오포스포라미데이트,이의 합성 및 용도
US6692918B2 (en) 1999-09-13 2004-02-17 Nugen Technologies, Inc. Methods and compositions for linear isothermal amplification of polynucleotide sequences
CA2384838C (fr) 1999-09-13 2006-07-18 Nugen Technologies, Inc. Procedes et compositions utiles pour l'amplification isothermique lineaire de sequences de polynucleotides
FR2798673B1 (fr) * 1999-09-16 2004-05-28 Exonhit Therapeutics Sa Methodes et compositions pour la detection d'evenements pathologiques
CA2398107C (fr) * 2000-01-28 2013-11-19 Althea Technologies, Inc. Procedes d'analyse de l'expression genique
US8076063B2 (en) * 2000-02-07 2011-12-13 Illumina, Inc. Multiplexed methylation detection methods
US6913884B2 (en) * 2001-08-16 2005-07-05 Illumina, Inc. Compositions and methods for repetitive use of genomic DNA
US20050214825A1 (en) * 2000-02-07 2005-09-29 John Stuelpnagel Multiplex sample analysis on universal arrays
US7955794B2 (en) 2000-09-21 2011-06-07 Illumina, Inc. Multiplex nucleic acid reactions
US7611869B2 (en) * 2000-02-07 2009-11-03 Illumina, Inc. Multiplexed methylation detection methods
US6770441B2 (en) * 2000-02-10 2004-08-03 Illumina, Inc. Array compositions and methods of making same
AU2001239760B2 (en) * 2000-02-10 2005-11-24 Illumina, Inc. Array of individual arrays as substrate for bead-based simultaneous processing of samples and manufacturing method therefor
DE10010281B4 (de) * 2000-02-25 2005-03-10 Epigenomics Ag Ligase/Polymerase-Verfahren zur Detektion von Cytosin-Methylierung in DNA Proben
US6368801B1 (en) 2000-04-12 2002-04-09 Molecular Staging, Inc. Detection and amplification of RNA using target-mediated ligation of DNA by RNA ligase
US7087414B2 (en) 2000-06-06 2006-08-08 Applera Corporation Methods and devices for multiplexing amplification reactions
ES2259666T3 (es) * 2000-06-21 2006-10-16 Bioarray Solutions Ltd Analisis molecular de multiples analitos usando series de particulas aleatorias con especificidad de aplicacion.
US7846733B2 (en) * 2000-06-26 2010-12-07 Nugen Technologies, Inc. Methods and compositions for transcription-based nucleic acid amplification
WO2002000938A2 (fr) 2000-06-26 2002-01-03 Nugen Technologies, Inc. Procedes et compositions permettant l'amplification d'acide nucleique fondee sur la transcription
JP2004501667A (ja) * 2000-07-01 2004-01-22 クロンディアグ　チップ　テヒノロギーズ　ゲーエムベーハー プローブ・アレイ上での分子相互作用の定性的および／または定量的検出方法
JP2004507230A (ja) * 2000-07-03 2004-03-11 アプレラ　コーポレイション ポリヌクレオチド配列アッセイ
BR0112163A (pt) * 2000-07-06 2004-02-10 Bio Merieux Processos de controle da qualidade microbiológica de um meio aquoso ambiental, kit de detecção microbiológica de um microorganismo presente em uma amostra e processo de produção e/ou desinfecção de um lìquido
US20050260574A1 (en) * 2000-07-27 2005-11-24 Gibbs Mark J Combinatorial probes and uses therefor
AU2001285155A1 (en) * 2000-08-21 2002-03-04 Lynx Therapeutics, Inc. Polymorphic dna fragments and uses thereof
EP1978110B1 (fr) * 2000-09-06 2010-05-26 Transnetyx, Inc. Procédé et système assistés par ordinateur pour criblage d'ADN genomique
US20050239125A1 (en) * 2000-09-06 2005-10-27 Hodge Timothy A Methods for genotype screening
US20050272085A1 (en) * 2000-09-06 2005-12-08 Hodge Timothy A Methods for forensic and congenic screening
US20030207289A1 (en) * 2001-09-04 2003-11-06 Hodge Timothy A. Detection of genetic sequences using a bipartite probe
US20040185464A1 (en) * 2000-09-15 2004-09-23 Kris Richard M. High throughput assay system
DE10048944A1 (de) * 2000-10-03 2002-04-18 Andreas Kage Verfahren zur Selektion hochaffin an ein Target bindender Nukleinsäuren durch zweidimensionale Auftrennung
US6858412B2 (en) * 2000-10-24 2005-02-22 The Board Of Trustees Of The Leland Stanford Junior University Direct multiplex characterization of genomic DNA
US20040018491A1 (en) * 2000-10-26 2004-01-29 Kevin Gunderson Detection of nucleic acid reactions on bead arrays
US20020142326A1 (en) * 2000-11-30 2002-10-03 Coull James M. Methods and compositions for sorting and/or determining organisms
WO2002048402A2 (fr) * 2000-12-13 2002-06-20 Nugen Technologies, Inc. Methodes et compositions de generation de copies multiples de sequences d'acides nucleiques et methodes de detection correspondantes
US6312929B1 (en) * 2000-12-22 2001-11-06 Cepheid Compositions and methods enabling a totally internally controlled amplification reaction
JP2004535769A (ja) * 2001-01-05 2004-12-02 インヴィトロジェン コーポレーション 結合した核酸の比較定量のための方法
FI115139B (fi) * 2001-01-10 2005-03-15 Valtion Teknillinen Menetelmä ja testipakkaus solu- tai kudosnäytteissä olevien polynukleotidien määrässä tapahtuvien vaihteluiden kvantitatiiviseen ja/tai vertailevaan arvioimiseen
WO2002064743A2 (fr) * 2001-02-12 2002-08-22 Rosetta Inpharmatics, Inc. Confirmation du contenu d'exon de transcrits d'arn par pcr au moyen d'amorces complementaires a chaque exon
WO2003012147A1 (fr) * 2001-02-20 2003-02-13 Datascope Investment Corp. Procede destine a reutiliser des transferts et des microreseaux classiques faisant appel a la technologie dendrimere adn
ZA200210369B (en) * 2001-03-09 2004-07-08 Nugen Technologies Inc Methods and compositions for amplification or RNA sequences.
US7094536B2 (en) * 2001-03-09 2006-08-22 Nugen Technologies, Inc. Methods and compositions for amplification of RNA sequences
US7029919B2 (en) * 2001-05-04 2006-04-18 Agilent Technologies, Inc. Electro-optical device and methods for hybridization and detection
WO2002090561A1 (fr) * 2001-05-07 2002-11-14 Curagen Corporation Procedes et compositions d'amplification d'acide nucleotide
US7838270B2 (en) * 2001-05-22 2010-11-23 The University Of Chicago Target-dependent transcription using deletion mutants of N4 RNA polymerase
GB0112868D0 (en) * 2001-05-25 2001-07-18 Secr Defence Detection system
DE10126630A1 (de) * 2001-05-31 2003-01-09 Peter Und Traudl Engelhorn Sti Verfahren zur Zellsortierung
CA2450139A1 (fr) * 2001-06-11 2002-12-19 Illumina, Inc Techniques de detection multiplexees
NZ530343A (en) * 2001-06-22 2007-01-26 Marshfield Clinic Methods and oligonucleotides for the detection of E. coli 0157:H7
US20030170695A1 (en) * 2001-06-29 2003-09-11 Liang Shi Enzymatic ligation-based identification of nucleotide sequences
US20030082584A1 (en) * 2001-06-29 2003-05-01 Liang Shi Enzymatic ligation-based identification of transcript expression
US7473767B2 (en) * 2001-07-03 2009-01-06 The Institute For Systems Biology Methods for detection and quantification of analytes in complex mixtures
EP1481076A4 (fr) * 2001-07-12 2005-05-11 Illumina Inc Reactions multiplex d'acides nucleiques
AU2002365115A1 (en) * 2001-07-20 2003-09-02 North Carolina State University Light addressable electrochemical detection of duplex structures
WO2003020983A1 (fr) * 2001-08-30 2003-03-13 Virginia Commonwealth University Pcr allele specifique pour genotypage
US20060014186A1 (en) * 2001-09-04 2006-01-19 Hodge Timothy A Methods for genotype screening of a strain disposed on an adsorbent carrier
US7153656B2 (en) * 2001-09-11 2006-12-26 Los Alamos National Security, Llc Nucleic acid sequence detection using multiplexed oligonucleotide PCR
JP2005504275A (ja) * 2001-09-18 2005-02-10 ユー．エス． ジェノミクス， インコーポレイテッド 高分解能線形解析用のポリマーの差示的タグ付け
JP2006500901A (ja) * 2001-09-26 2006-01-12 エピジェンクス ファーマスーティカルズ、 インコーポレイテッド Ｄｎａメチル化変化のアッセイ
US20080138800A1 (en) * 2001-10-15 2008-06-12 Alice Xiang Li Multiplexed analysis of polymorphic loci by concurrent interrogation and enzyme-mediated detection
JP3829690B2 (ja) * 2001-10-30 2006-10-04 株式会社日立製作所 核酸配列の検査方法
WO2003054167A2 (fr) * 2001-12-20 2003-07-03 Merck & Co., Inc. Identification de sites polymorphes dans le gene humain mglur8 et utilisations associees
EP1327682B1 (fr) * 2002-01-11 2009-05-13 BioSpring Gesellschaft für Biotechnologie mbH Procédé de production de l'ADN
US20050208491A1 (en) * 2002-02-08 2005-09-22 Rudolf Zirwes Specific multiplex analysis of nucleic acids
US7499806B2 (en) 2002-02-14 2009-03-03 Illumina, Inc. Image processing in microsphere arrays
AU2003220254B2 (en) * 2002-03-13 2008-09-18 Syngenta Participations, Ag. Nucleic acid detection method
US7081339B2 (en) * 2002-04-12 2006-07-25 Primera Biosystems, Inc. Methods for variation detection
DE10220935B3 (de) * 2002-05-10 2004-02-05 Siemens Ag Verfahren für die biochemische Analytik von DNA und zugehörige Anordnung
JP2005527219A (ja) * 2002-05-24 2005-09-15 シジェン インコーポレイテッド デオキシリボ核酸の平行二重鎖及びその使用方法
EP1546380A4 (fr) * 2002-05-28 2007-02-14 Us Genomics Inc Procedes et appareils d'analyse de polymeres simples
DK1512015T3 (da) * 2002-06-12 2009-07-06 Genencor Int Fremgangsmåder til forbedring af bindingsegenskaber hos et molekyle
FI20021325A0 (fi) * 2002-07-05 2002-07-05 Valtion Teknillinen Menetelmä ja reagenssipakkaus yksittäisten polynukleotidien määrän määrittämiseksi
US7192700B2 (en) * 2002-12-20 2007-03-20 Orchid Cellmark Inc. Methods and compositions for conducting primer extension and polymorphism detection reactions
JP2005538735A (ja) * 2002-09-17 2005-12-22 パーキネルマー ラス インコーポレイテッド 核酸反応のリアルタイム検出法
JP2006500034A (ja) * 2002-09-19 2006-01-05 アプレラ コーポレイション 標的を検出するための方法および組成物
US20040214196A1 (en) * 2002-09-19 2004-10-28 Applera Corporation Methods and compositions for detecting targets
US20040259105A1 (en) * 2002-10-03 2004-12-23 Jian-Bing Fan Multiplex nucleic acid analysis using archived or fixed samples
GB0223563D0 (en) * 2002-10-10 2002-11-20 Secr Defence Detection system
US20040235005A1 (en) * 2002-10-23 2004-11-25 Ernest Friedlander Methods and composition for detecting targets
EP1558756A4 (fr) * 2002-10-23 2006-09-27 Applera Corp Procedes et composition de detection de cibles
WO2004046344A2 (fr) * 2002-11-19 2004-06-03 Applera Corporation Dosage de detection de sequences polynucleotidiques
WO2004046343A2 (fr) * 2002-11-19 2004-06-03 Applera Corporation Methodes de detection et analyse de sequences polynucleotidiques
GB2395557A (en) * 2002-11-22 2004-05-26 Dynal Biotech Ltd Nucleic acid probes
AU2003298706A1 (en) 2002-12-04 2004-06-23 Applera Corporation Multiplex amplification of polynucleotides
WO2004053105A2 (fr) * 2002-12-12 2004-06-24 Nanosphere, Inc. Detection de snp directe avec adn non amplifie
US8206904B2 (en) 2002-12-18 2012-06-26 Third Wave Technologies, Inc. Detection of nucleic acids
US7851150B2 (en) 2002-12-18 2010-12-14 Third Wave Technologies, Inc. Detection of small nucleic acids
CA2521999A1 (fr) 2002-12-20 2004-09-02 Biotrove, Inc. Appareil et procede de dosage utilisant des reseaux microfluidiques
US20060094108A1 (en) * 2002-12-20 2006-05-04 Karl Yoder Thermal cycler for microfluidic array assays
US9487823B2 (en) * 2002-12-20 2016-11-08 Qiagen Gmbh Nucleic acid amplification
CA2511012C (fr) 2002-12-20 2018-02-27 Applera Corporation Polymorphismes genetiques associes a l'infarctus du myocarde, techniques de detection et utilisations de ceux-ci
US20040180369A1 (en) * 2003-01-16 2004-09-16 North Carolina State University Photothermal detection of nucleic acid hybridization
US8043834B2 (en) * 2003-03-31 2011-10-25 Qiagen Gmbh Universal reagents for rolling circle amplification and methods of use
WO2004092418A2 (fr) * 2003-04-14 2004-10-28 Nugen Technologies, Inc. Amplification globale effectuee avec une amorce composite amorcee de maniere aleatoire
WO2004094653A2 (fr) * 2003-04-22 2004-11-04 Irm Llc Analyse differentielle de la longueur des marqueurs de la proliferation cellulaire
US20040229269A1 (en) * 2003-05-15 2004-11-18 Ghazala Hashmi Hybridization-mediated analysis of polymorphisms
DE10323685A1 (de) * 2003-05-22 2004-12-09 Rühe, Jürgen, Prof. Dr. Verfahren zur kovalenten Immobilisierung von Sonden-Biomolekülen an organischen Oberflächen
ES2284021T3 (es) * 2003-06-02 2007-11-01 Check-Points Holding B.V. Procedimiento rapido para detectar microorganismos en muestras de alimentos.
US20050239089A1 (en) * 2003-06-06 2005-10-27 Johnson Martin D Mobility cassettes
WO2005001113A2 (fr) * 2003-06-27 2005-01-06 Thomas Jefferson University Procedes de detection de variations d'acides nucleiques
KR100683025B1 (ko) * 2003-06-30 2007-02-15 마츠시타 덴끼 산교 가부시키가이샤 뉴클레오티드쇄 수식방법
US20100291637A1 (en) * 2003-06-30 2010-11-18 Panasonic Corporation Method for modifying nucleotide chain
CN1580283A (zh) * 2003-08-13 2005-02-16 清华大学 一种检测核酸分子的方法
GB0319949D0 (en) * 2003-08-26 2003-09-24 Univ Strathclyde Nucleic acid sequence identification
US20050266417A1 (en) * 2003-09-12 2005-12-01 Francis Barany Methods for identifying target nucleic acid molecules
WO2005029705A2 (fr) * 2003-09-18 2005-03-31 Bioarray Solutions, Ltd. Codage numerique pour identification de sous-types des types de supports solides codes
US20050089916A1 (en) * 2003-10-28 2005-04-28 Xiongwu Xia Allele assignment and probe selection in multiplexed assays of polymorphic targets
ES2533876T3 (es) 2003-10-29 2015-04-15 Bioarray Solutions Ltd Análisis multiplexado de ácidos nucleicos mediante fragmentación de ADN bicatenario
WO2005042785A1 (fr) * 2003-10-30 2005-05-12 North Carolina State University Detection electrochimique d'hybridation d'acide nucleique
US20050191651A1 (en) * 2003-10-30 2005-09-01 North Carolina State University Temperature-jump enhanced electrochemical detection of nucleic acid hybridization
CA3050151C (fr) 2003-11-26 2023-03-07 Celera Corporation Polymorphismes nucleotides simples associes a des troubles cardiovasculaires et a une reponse au medicament, leurs procedes de detection et d'utilisation
US20050130213A1 (en) * 2003-12-10 2005-06-16 Tom Morrison Selective ligation and amplification assay
US7582430B2 (en) * 2004-01-20 2009-09-01 United States Of America As Represented By The Secretary Of The Army Immunoliposome-nucleic acid amplification (ILNAA) assay
EP1561823A1 (fr) * 2004-02-04 2005-08-10 Biotez Berlin-Buch GmbH Procédé pour la détection des polymorphismes de nucléotide simple (SNP) des gênes du métabolisme des médicaments et dispositif pour l'utilisation correspondante
EP1715954A1 (fr) * 2004-02-18 2006-11-02 Applera Corporation Essais biologiques a etapes multiples sur des plates-formes d'applications micro-fluidiques modulaires
EP1753873A4 (fr) * 2004-03-05 2008-11-05 Ohio Med College Procedes et compositions pour l'evaluation d'acides nucleiques et d'alleles
ATE399884T1 (de) * 2004-03-24 2008-07-15 Applera Corp Codierungs- und decodierungsreaktionen zur bestimmung von target-polynukleotiden
CA2568238C (fr) * 2004-04-12 2013-10-15 Medical College Of Ohio Methodes et compositions pour analyser des analytes
WO2005108618A2 (fr) * 2004-04-30 2005-11-17 Applera Corporation Procedes et kits de detection de methylation
EP1745154B1 (fr) 2004-05-07 2012-08-01 Celera Corporation Polymorphismes genetiques associes a des procedes de detection de fibrose du foie et utilisations de ceux-ci
US7622281B2 (en) * 2004-05-20 2009-11-24 The Board Of Trustees Of The Leland Stanford Junior University Methods and compositions for clonal amplification of nucleic acid
US7575863B2 (en) 2004-05-28 2009-08-18 Applied Biosystems, Llc Methods, compositions, and kits comprising linker probes for quantifying polynucleotides
WO2005118871A1 (fr) * 2004-05-28 2005-12-15 The Arizona Board Of Regents Capteur par resonance plasmonique de surface permettant de detecter des modifications dans la masse de polynucleotides
US7785843B2 (en) * 2004-06-23 2010-08-31 Sequenom, Inc. Target-specific compomers and methods of use
US7363170B2 (en) * 2004-07-09 2008-04-22 Bio Array Solutions Ltd. Transfusion registry network providing real-time interaction between users and providers of genetically characterized blood products
CN101087890A (zh) * 2004-07-26 2007-12-12 帕拉列勒生物科学公司 多个基因组的同时分析
DE112005001815A5 (de) * 2004-07-30 2007-06-14 Universität Bremen Verfahren zum Analysieren von Proben mittels einer Hybridisierung
DE102004037081A1 (de) * 2004-07-30 2006-03-23 Universität Bremen Verfahren zum Analysieren von Proben mittels einer Hybridisierung
EP1623996A1 (fr) * 2004-08-06 2006-02-08 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Procédé amélioré de selection d'une protéine desirée d'une bibliothéque.
US20060040258A1 (en) * 2004-08-23 2006-02-23 Huiyan Guo Water-soluble conjugates and methods of preparation
US7642055B2 (en) 2004-09-21 2010-01-05 Applied Biosystems, Llc Two-color real-time/end-point quantitation of microRNAs (miRNAs)
ES2301268B1 (es) * 2004-10-25 2009-05-01 Centro De Investigacion Biomolecular Aplicada Salamanca, S.L. Empleo del gen slug, o de sus productos de replicacion, transcripcion o expresion, en la identificacion, diagnostico, prevencion o tratamiento de la diseminacion del cancer y/o desarrollo de metastasis.
WO2006059769A1 (fr) * 2004-12-03 2006-06-08 Aichi Prefecture Procede de diagnostic de lymphome malin et d'estimation du pronostic associe
US20060134650A1 (en) * 2004-12-21 2006-06-22 Illumina, Inc. Methylation-sensitive restriction enzyme endonuclease method of whole genome methylation analysis
EP1831401B1 (fr) * 2004-12-29 2010-02-10 Applied Biosystems, LLC Procedes, compositions et kits pour former des polynucleotides auto-complementaires
US20060211024A1 (en) * 2005-03-10 2006-09-21 Gwc Technologies Incorporated Methods for analysis of a nucleic acid sample
EP1856296A2 (fr) 2005-03-11 2007-11-21 Applera Corporation Polymorphismes genetiques associes a des maladies cardiaques coronariennes, methodes de detection et d'utilisation desdits polymorphismes
US20060205090A1 (en) * 2005-03-14 2006-09-14 Newton Michael W Water-soluble conjugates for electrochemical detection
US20070026423A1 (en) * 2005-03-16 2007-02-01 Thomas Koehler Method and test kit for the detection of target nucleic acids
US8309303B2 (en) * 2005-04-01 2012-11-13 Qiagen Gmbh Reverse transcription and amplification of RNA with simultaneous degradation of DNA
US20060246463A1 (en) * 2005-04-20 2006-11-02 Vevea Dirk N Methods and oligonucleotides for the detection of Salmonella SP., E coli 0157:H7, and Listeria monocytogenes
US20060240442A1 (en) * 2005-04-20 2006-10-26 Vevea Dirk N Methods and oligonucleotides for the detection of Salmonella SP., E coli 0157:H7, and Listeria monocytogenes
US20070009925A1 (en) * 2005-05-05 2007-01-11 Applera Corporation Genomic dna sequencing methods and kits
WO2007117256A1 (fr) * 2005-05-31 2007-10-18 Applera Corporation Amplification multiplexée d'acides nucléiques courts
US8486629B2 (en) 2005-06-01 2013-07-16 Bioarray Solutions, Ltd. Creation of functionalized microparticle libraries by oligonucleotide ligation or elongation
US20060276220A1 (en) * 2005-06-03 2006-12-07 Judith Schure Cell phone case with magnifier and method
US20070065844A1 (en) * 2005-06-08 2007-03-22 Massachusetts Institute Of Technology Solution-based methods for RNA expression profiling
US20070087360A1 (en) * 2005-06-20 2007-04-19 Boyd Victoria L Methods and compositions for detecting nucleotides
DE602006018622D1 (de) * 2005-06-30 2011-01-13 Ge Healthcare Bio Sciences Nachweisverfahren für die genexpression
US7977108B2 (en) * 2005-07-25 2011-07-12 Roche Molecular Systems, Inc. Method for detecting a mutation in a repetitive nucleic acid sequence
US20070065847A1 (en) * 2005-08-11 2007-03-22 Affymetrix, Inc. Degeneratively Labeled Probes
WO2007025281A2 (fr) 2005-08-24 2007-03-01 Applera Corporation Procede permettant de quantifier des arnsi, arnmi et des arnmi polymorphes
CA2621267A1 (fr) 2005-09-07 2007-03-15 Nugen Technologies, Inc. Procedure d'amplication d'acide nucleique amelioree
EP1762627A1 (fr) 2005-09-09 2007-03-14 Qiagen GmbH Procédé pour l'activation d'acides nucléiques pour effectuer une réaction d'une polymérase
ATE389035T1 (de) * 2005-09-13 2008-03-15 Eppendorf Array Tech Sa Verfahren zum nachweis von homologen sequenzen, welche sich durch eine base unterscheiden, auf einem mikroarray
US7799530B2 (en) 2005-09-23 2010-09-21 Celera Corporation Genetic polymorphisms associated with cardiovascular disorders and drug response, methods of detection and uses thereof
US20070141604A1 (en) * 2005-11-15 2007-06-21 Gormley Niall A Method of target enrichment
WO2007065025A2 (fr) * 2005-11-29 2007-06-07 Wisconsin Alumni Research Foundation Procede d'analyse d'adn au moyen de micro/nano-canaux
US7932037B2 (en) * 2007-12-05 2011-04-26 Perkinelmer Health Sciences, Inc. DNA assays using amplicon probes on encoded particles
US20090104613A1 (en) * 2005-12-23 2009-04-23 Perkinelmer Las, Inc. Methods and compositions relating to multiplexed genomic gain and loss assays
CA2640385C (fr) 2005-12-23 2014-07-15 Nanostring Technologies, Inc. Nanorapporteurs et procedes de production et d'utilisation de ceux-ci
BRPI0620420B1 (pt) * 2005-12-23 2016-08-09 Perkinelmer Las Inc método para avaliar dna genômico
US20100009373A1 (en) * 2005-12-23 2010-01-14 Perkinelmer Health Sciences, Inc. Methods and compositions relating to multiplex genomic gain and loss assays
SG10201806098QA (en) 2006-02-13 2018-08-30 Us Gov Health & Human Services Primers and probes for detection and discrimination of types and subtypes of influenza viruses
WO2007101075A2 (fr) * 2006-02-22 2007-09-07 Applera Corporation Procédé de double-ligature pour détecter le polymorphisme haplotype et grande échelle
US20070196832A1 (en) * 2006-02-22 2007-08-23 Efcavitch J William Methods for mutation detection
CN101395280A (zh) * 2006-03-01 2009-03-25 凯津公司 基于测序的高通量SNPs连接检测技术
US8673567B2 (en) * 2006-03-08 2014-03-18 Atila Biosystems, Inc. Method and kit for nucleic acid sequence detection
US10522240B2 (en) 2006-05-03 2019-12-31 Population Bio, Inc. Evaluating genetic disorders
US7702468B2 (en) 2006-05-03 2010-04-20 Population Diagnostics, Inc. Evaluating genetic disorders
DE102006020885A1 (de) * 2006-05-05 2007-11-08 Qiagen Gmbh Einführung von Sequenzelementen in Nukleinsäuren
EP2530168B1 (fr) 2006-05-11 2015-09-16 Raindance Technologies, Inc. Dispositifs microfluidiques
US7833716B2 (en) 2006-06-06 2010-11-16 Gen-Probe Incorporated Tagged oligonucleotides and their use in nucleic acid amplification methods
US20100056388A1 (en) * 2006-08-21 2010-03-04 Cnvgenes, Inc. Nucleic acid array having fixed nucleic acid anti-probes and complementary free nucleic acid probes
US20090286694A1 (en) * 2006-08-21 2009-11-19 Gafur Zainiev Nucleic acid array with releaseable nucleic acid probes
US20080044821A1 (en) * 2006-08-21 2008-02-21 Gafur Zainiev Nucleic acid array having fixed nucleic acid anti-probes and complementary free nucleic acid probes
US20080044822A1 (en) * 2006-08-21 2008-02-21 Gafur Zainiev Nucleic acid array with releaseable nucleic acid probes
US20080050724A1 (en) * 2006-08-24 2008-02-28 Microfluidic Systems, Inc. Method of detecting one or more limited copy targets
JP2008072950A (ja) * 2006-09-21 2008-04-03 Sysmex Corp 変換処理の確認方法及びこれに用いられる核酸分子
EP2076609A1 (fr) 2006-10-10 2009-07-08 Illumina Inc. Compositions et procédés pour une sélection représentative d'acides nucléiques à partir de mélanges complexes
US20080090238A1 (en) * 2006-10-12 2008-04-17 Dan-Hui Dorothy Yang Increased sensitivity of proximity ligation assays
CA2666346C (fr) 2006-10-20 2016-02-23 Celera Corporation Polymorphismes genetiques associes a la thrombose veineuse, procedes pour les detecter et utilisations
US20090074637A1 (en) * 2006-11-03 2009-03-19 Murphy Michael C Optimized Modular Microfluidic Devices
US8133701B2 (en) * 2006-12-05 2012-03-13 Sequenom, Inc. Detection and quantification of biomolecules using mass spectrometry
US7902345B2 (en) * 2006-12-05 2011-03-08 Sequenom, Inc. Detection and quantification of biomolecules using mass spectrometry
JP2008148570A (ja) * 2006-12-14 2008-07-03 Hitachi Ltd 微生物検出システム
JP5340167B2 (ja) * 2006-12-21 2013-11-13 ジェン−プロウブ インコーポレイテッド 核酸増幅のための方法および組成物
NZ619576A (en) 2006-12-27 2014-07-25 Harvard College Compositions and methods for the treatment of infections and tumors
US8930178B2 (en) 2007-01-04 2015-01-06 Children's Hospital Medical Center Processing text with domain-specific spreading activation methods
US20080182235A1 (en) * 2007-01-30 2008-07-31 Celsis International Plc Detection of Analytes in Samples Using Liposome-Amplified Luminescence and Magnetic Separation
WO2008097559A2 (fr) 2007-02-06 2008-08-14 Brandeis University Manipulation de fluides et de réactions dans des systèmes microfluidiques
JP2010520745A (ja) 2007-02-07 2010-06-17 デコード・ジェネティクス・イーエイチエフ 前立腺癌のリスクに寄与する遺伝子変異体
US8906620B2 (en) 2007-03-23 2014-12-09 Dana-Farber Cancer Institute, Inc. Exon grouping analysis
WO2008130623A1 (fr) 2007-04-19 2008-10-30 Brandeis University Manipulation de fluides, composants fluidiques et réactions dans des systèmes microfluidiques
US20080274458A1 (en) * 2007-05-01 2008-11-06 Latham Gary J Nucleic acid quantitation methods
CA2690608C (fr) 2007-05-21 2020-07-21 Genentech, Inc. Procedes et compositions pour l'identification et le traitement du lupus
US20090036325A1 (en) * 2007-05-25 2009-02-05 Applera Corporation Directed assembly of amplicons to enhance read pairing signature with massively parallel short read sequencers
MX2009012722A (es) 2007-05-25 2009-12-11 Decode Genetics Ehf Variantes geneticas sobre chr 5p12 y 10q26 como marcadores para el uso en la evaluacion del riesgo, diagnostico, pronostico y tratamiento del cancer de mama.
US7947446B2 (en) * 2007-05-29 2011-05-24 Ming-Sheng Lee High throughput mutation screening methods and kits using a universalized approach—differential sequence fill-in (DSF)-enabled sequential adapter ligation and amplification
EP2527472B1 (fr) 2007-05-31 2016-09-28 Yale University Une lésion génétique associée à un cancer
JP5730568B2 (ja) * 2007-06-01 2015-06-10 モノクアント・ピーティーワイ・リミテッド Ｄｎａ切断点の分析のための方法
WO2008154085A1 (fr) * 2007-06-06 2008-12-18 Bio-Rad Laboratories, Inc. Amplification de signal employant des sondes en épingle à cheveux circulaires
JP5165933B2 (ja) * 2007-06-12 2013-03-21 日本碍子株式会社 標的核酸中の特定部分配列の検出方法及びアレイ
US8008010B1 (en) * 2007-06-27 2011-08-30 Applied Biosystems, Llc Chimeric oligonucleotides for ligation-enhanced nucleic acid detection, methods and compositions therefor
US9512470B2 (en) * 2007-07-11 2016-12-06 Pathofinder Holding B.V. Method for the simultaneous detection of multiple nucleic acid sequences in a sample
EP2195452B1 (fr) 2007-08-29 2012-03-14 Sequenom, Inc. Méthodes et compositions s'appliquant à une amplification en chaîne par polymèrase universelle spécifique de la taille
WO2009036525A2 (fr) * 2007-09-21 2009-03-26 Katholieke Universiteit Leuven Outils et procédés pour tests génétiques ayant recours à un séquençage de dernière génération
EA201000427A1 (ru) * 2007-10-04 2010-10-29 Хэлсион Молекулар Секвенирование нуклеиново-кислотных полимеров с использованием электронной микроскопии
CA2702169A1 (fr) * 2007-10-12 2009-04-16 Decode Genetics Ehf Variants de sequence pour deduire des motifs de pigmentation humaine
US7759112B2 (en) * 2007-10-31 2010-07-20 Akonni Biosystems, Inc. Apparatus, system, and method for purifying nucleic acids
US10125388B2 (en) 2007-10-31 2018-11-13 Akonni Biosystems, Inc. Integrated sample processing system
US20090111193A1 (en) 2007-10-31 2009-04-30 Cooney Christopher G Sample preparation device
US9428746B2 (en) 2007-10-31 2016-08-30 Akonni Biosystems, Inc. Method and kit for purifying nucleic acids
US8039212B2 (en) 2007-11-05 2011-10-18 Celera Corporation Genetic polymorphisms associated with liver fibrosis, methods of detection and uses thereof
WO2009073629A2 (fr) 2007-11-29 2009-06-11 Complete Genomics, Inc. Procédés de séquençage aléatoire efficace
US8093063B2 (en) * 2007-11-29 2012-01-10 Quest Diagnostics Investments Incorporated Assay for detecting genetic abnormalities in genomic nucleic acids
US8697360B2 (en) 2007-11-30 2014-04-15 Decode Genetics Ehf. Genetic variants on CHR 11Q and 6Q as markers for prostate and colorectal cancer predisposition
US20090181390A1 (en) * 2008-01-11 2009-07-16 Signosis, Inc. A California Corporation High throughput detection of micrornas and use for disease diagnosis
AU2009205523A1 (en) 2008-01-14 2009-07-23 Applied Biosystems, Llc Compositions, methods, and kits for detecting ribonucleic acid
US8034568B2 (en) * 2008-02-12 2011-10-11 Nugen Technologies, Inc. Isothermal nucleic acid amplification methods and compositions
CA2714521A1 (fr) * 2008-02-14 2009-08-20 Decode Genetics Ehf Variants de sensibilite pour le cancer du poumon
US20090221620A1 (en) 2008-02-20 2009-09-03 Celera Corporation Gentic polymorphisms associated with stroke, methods of detection and uses thereof
US20090253142A1 (en) * 2008-03-15 2009-10-08 Hologic, Inc. Compositions and methods for analysis of nucleic acid molecules during amplification reactions
GB2470672B (en) 2008-03-21 2012-09-12 Nugen Technologies Inc Methods of RNA amplification in the presence of DNA
JP5319148B2 (ja) * 2008-03-27 2013-10-16 日本碍子株式会社 標的核酸中の変異の検出方法及びアレイ
CA2724666A1 (fr) 2008-04-01 2009-10-08 Decode Genetics Ehf Variants de susceptibilite a une maladie arterielle peripherique et un anevrisme aortique abdominal
PT2271767T (pt) * 2008-04-03 2016-08-18 Cb Biotechnologies Inc Reação em cadeia de polimerase para captura de amplicões em multiplexo para amplificação de alvos múltiplos
EP2281198B1 (fr) * 2008-04-19 2013-01-02 New York University Peptide de mycobacterium tuberculosis immunodominants provenant de protéines de paroi cellulaire pour un diagnostic précoce et une immunisation
JP2009268665A (ja) * 2008-05-07 2009-11-19 Canon Inc 吸入装置
AU2009246180B2 (en) 2008-05-14 2015-11-05 Dermtech International Diagnosis of melanoma and solar lentigo by nucleic acid analysis
CN101586150B (zh) * 2008-05-23 2016-09-28 陕西佰美基因股份有限公司 检测探针、通用寡核苷酸芯片及核酸检测方法及其用途
WO2010001419A2 (fr) * 2008-07-04 2010-01-07 Decode Genetics Ehf Variations du nombre de copies prédictives d’un risque de schizophrénie
CA2729934A1 (fr) 2008-07-07 2010-01-14 Decode Genetics Ehf Variantes genetiques pour l'evaluation du risque de cancer du sein
EP2878680B1 (fr) 2008-07-09 2016-06-08 Celera Corporation Polymorphismes génétiques associés à des maladies cardiovasculaires, procédés de détection et utilisations associées
EP4047367A1 (fr) 2008-07-18 2022-08-24 Bio-Rad Laboratories, Inc. Procedé de détection d'analytes cibles au moyens des bibliothèques de gouttelettes
WO2010012002A1 (fr) * 2008-07-25 2010-01-28 Saryna Medical Corporation Procédés et systèmes pour l'analyse génétique des érythrocytes nucléés fœtaux
JP5272230B2 (ja) * 2008-07-30 2013-08-28 日鉄住金環境株式会社 ユニバーサルな核酸プローブセットおよびその使用方法
CN102177252B (zh) * 2008-08-12 2014-12-24 解码遗传学私营有限责任公司 用于甲状腺癌症的风险评估的遗传变型
US20110212855A1 (en) * 2008-08-15 2011-09-01 Decode Genetics Ehf. Genetic Variants Predictive of Cancer Risk
EP2326732A4 (fr) * 2008-08-26 2012-11-14 Fluidigm Corp Procédés de dosage pour un débit amélioré d'échantillons et/ou de cibles
CN102282265B (zh) 2008-11-28 2020-07-24 埃默里大学 用于治疗传染病和肿瘤的方法
WO2010077324A2 (fr) 2008-12-17 2010-07-08 Life Technologies Corporation Procédés, compositions et nécessaires de détection de variantes alléliques
SG173081A1 (en) 2009-01-30 2011-08-29 Kantonsspital Aarau Ag Gene dosage analysis
US20120028254A1 (en) 2009-02-06 2012-02-02 Weidhaas Joanne B SNP Marker of Breast and Ovarian Cancer Risk
WO2010096713A2 (fr) 2009-02-20 2010-08-26 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Procédé pour le diagnostic de troubles vasculaires liés à l'âge
JP5805064B2 (ja) 2009-03-27 2015-11-04 ライフ テクノロジーズ コーポレーション 対立遺伝子変種を検出するための方法、組成物、およびキット
EP2414541B8 (fr) * 2009-03-31 2014-09-10 Centre Hospitalier Universitaire Vaudois Macropuce pour l'identification de méthylations dépendante d'une ligature (mlm)
EP2765205B1 (fr) * 2009-04-01 2016-05-18 DxTerity Diagnostics Incorporated Amplification de sonde dépendante de ligature chimique (CLPA)
CN102439177B (zh) 2009-04-02 2014-10-01 弗卢伊蒂格姆公司 用于对目标核酸进行条形码化的多引物扩增方法
CN102449165B (zh) 2009-04-03 2014-07-09 解码遗传学私营有限责任公司 用于心房颤动和中风的风险管理的遗传标志
EP2256215A1 (fr) 2009-04-30 2010-12-01 Steffen Mergemeier Système d'analyse utilisant une activité de nucléase d'une polymérase d'acide nucléique
US20110003301A1 (en) * 2009-05-08 2011-01-06 Life Technologies Corporation Methods for detecting genetic variations in dna samples
NZ596070A (en) * 2009-05-08 2013-10-25 Decode Genetics Ehf Genetic variants contributing to risk of prostate cancer
US20100299773A1 (en) * 2009-05-20 2010-11-25 Monsanto Technology Llc Methods and compositions for selecting an improved plant
WO2010138926A1 (fr) 2009-05-29 2010-12-02 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, Centers For Disease Control And Prevention Acides nucléiques pour la détection et la discrimination des génotypes de chlamydophila psittaci
AU2010253978B2 (en) 2009-05-29 2014-12-11 Ventana Medical Systems, Inc. Methods of scoring gene copy number in a biological sample using in situ hybridization
WO2010138908A1 (fr) 2009-05-29 2010-12-02 Ventana Medical Systems, Inc. Nombre de copies du gène igf1r en tant que marqueur pronostique dans un cancer du poumon non à petites cellules
EP2446056A2 (fr) 2009-06-25 2012-05-02 Yale University Single-nucleotide polymorphism dans brca1 et risque pour cancer
US9169512B2 (en) 2009-07-01 2015-10-27 Gen-Probe Incorporated Methods and compositions for nucleic acid amplification
EP2451977A4 (fr) 2009-07-10 2013-01-02 Decode Genetics Ehf Marqueurs génétiques associés au risque de diabète sucré
US9487839B2 (en) * 2009-09-29 2016-11-08 Case Western Reserve University Method for detecting single nucleotide polymorphisms
WO2011044205A1 (fr) 2009-10-07 2011-04-14 Genentech, Inc. Procédés pour traiter, diagnostiquer et surveiller un lupus
WO2011052586A1 (fr) * 2009-10-29 2011-05-05 日本碍子株式会社 Procédé de détection d'un acide nucléique cible
EP2510126B1 (fr) * 2009-12-07 2017-08-09 Illumina, Inc. Indexage multi-échantillon pour un génotypage multiplexe
US8835358B2 (en) 2009-12-15 2014-09-16 Cellular Research, Inc. Digital counting of individual molecules by stochastic attachment of diverse labels
EP2526207A4 (fr) 2010-01-22 2013-10-09 Univ California Compositions et procédés de criblage chimique à base de signature génique
EP2534267B1 (fr) 2010-02-12 2018-04-11 Raindance Technologies, Inc. Analyse numérique d'analytes
US9399797B2 (en) 2010-02-12 2016-07-26 Raindance Technologies, Inc. Digital analyte analysis
US8774488B2 (en) 2010-03-11 2014-07-08 Cellscape Corporation Method and device for identification of nucleated red blood cells from a maternal blood sample
US20190300945A1 (en) 2010-04-05 2019-10-03 Prognosys Biosciences, Inc. Spatially Encoded Biological Assays
US10787701B2 (en) 2010-04-05 2020-09-29 Prognosys Biosciences, Inc. Spatially encoded biological assays
JP5893607B2 (ja) 2010-04-05 2016-03-23 プログノシス バイオサイエンシズ インコーポレイテッドＰｒｏｇｎｏｓｙｓ Ｂｉｏｓｃｉｅｎｃｅｓ，Ｉｎｃ． 空間コード化生物学的アッセイ
CA2796509C (fr) 2010-04-16 2019-04-09 Jonas M. Winchell Dosage par pcr en temps reel pour detecter des pathogenes respiratoires bacteriens
US9828637B2 (en) 2010-04-18 2017-11-28 Wake Forest University Health Sciences Methods of predicting predisposition to or risk of kidney disease
US20110269735A1 (en) 2010-04-19 2011-11-03 Celera Corporation Genetic polymorphisms associated with statin response and cardiovascular diseases, methods of detection and uses thereof
US20130316338A1 (en) 2010-06-29 2013-11-28 The United States Government As Represented By The Department Of Veterans Affairs CCR6 As A Biomarker of Alzheimer's Disease
US8586301B2 (en) 2010-06-30 2013-11-19 Stratos Genomics, Inc. Multiplexed identification of nucleic acid sequences
WO2012018387A2 (fr) 2010-08-02 2012-02-09 Population Diagnotics, Inc. Compositions et méthodes de recherche de mutations causales dans des troubles génétiques
US20130261003A1 (en) 2010-08-06 2013-10-03 Ariosa Diagnostics, In. Ligation-based detection of genetic variants
US8700338B2 (en) 2011-01-25 2014-04-15 Ariosa Diagnosis, Inc. Risk calculation for evaluation of fetal aneuploidy
US11203786B2 (en) 2010-08-06 2021-12-21 Ariosa Diagnostics, Inc. Detection of target nucleic acids using hybridization
US11031095B2 (en) 2010-08-06 2021-06-08 Ariosa Diagnostics, Inc. Assay systems for determination of fetal copy number variation
US20140342940A1 (en) 2011-01-25 2014-11-20 Ariosa Diagnostics, Inc. Detection of Target Nucleic Acids using Hybridization
US10533223B2 (en) 2010-08-06 2020-01-14 Ariosa Diagnostics, Inc. Detection of target nucleic acids using hybridization
US20130040375A1 (en) 2011-08-08 2013-02-14 Tandem Diagnotics, Inc. Assay systems for genetic analysis
US20120034603A1 (en) 2010-08-06 2012-02-09 Tandem Diagnostics, Inc. Ligation-based detection of genetic variants
US10167508B2 (en) 2010-08-06 2019-01-01 Ariosa Diagnostics, Inc. Detection of genetic abnormalities
CN103221543B (zh) 2010-08-30 2016-10-05 陶氏益农公司 用于玉米的激活标签化平台和所得的带标签的群体和植物
PL2611925T3 (pl) 2010-08-30 2018-05-30 Dow Agrosciences, Llc Wzmacniacz z bacilokształtnego wirusa trzciny cukrowej (SCBV) i jego zastosowanie w genomice funkcjonalnej roślin
WO2012034013A2 (fr) 2010-09-10 2012-03-15 Bio-Rad Laboratories, Inc. Détection des régions d'interaction de l'arn au sein de l'adn
US9353406B2 (en) 2010-10-22 2016-05-31 Fluidigm Corporation Universal probe assay methods
US20120108651A1 (en) 2010-11-02 2012-05-03 Leiden University Medical Center (LUMC) Acting on Behalf of Academic Hospital Leiden (AZL) Genetic polymorphisms associated with venous thrombosis and statin response, methods of detection and uses thereof
US8932868B2 (en) * 2010-12-08 2015-01-13 Imec Biosensor using impedimetric real-time monitoring
WO2012095872A1 (fr) 2011-01-13 2012-07-19 Decode Genetics Ehf Variants génétiques comme marqueurs à utiliser dans l'évaluation du risque, le diagnostic, le pronostic et le traitement du cancer de la vessie
EP3216878B1 (fr) 2011-01-17 2019-04-03 Life Technologies Corporation Workflow pour la détection de ligands à l'aide d'acides nucléiques
SG192108A1 (en) 2011-01-25 2013-08-30 Almac Diagnostics Ltd Colon cancer gene expression signatures and methods of use
US10131947B2 (en) 2011-01-25 2018-11-20 Ariosa Diagnostics, Inc. Noninvasive detection of fetal aneuploidy in egg donor pregnancies
US20120190020A1 (en) 2011-01-25 2012-07-26 Aria Diagnostics, Inc. Detection of genetic abnormalities
US9994897B2 (en) 2013-03-08 2018-06-12 Ariosa Diagnostics, Inc. Non-invasive fetal sex determination
US11270781B2 (en) 2011-01-25 2022-03-08 Ariosa Diagnostics, Inc. Statistical analysis for non-invasive sex chromosome aneuploidy determination
US8756020B2 (en) 2011-01-25 2014-06-17 Ariosa Diagnostics, Inc. Enhanced risk probabilities using biomolecule estimations
US20120208193A1 (en) 2011-02-15 2012-08-16 Bio-Rad Laboratories, Inc. Detecting methylation in a subpopulation of genomic dna
EP2675914A1 (fr) 2011-02-18 2013-12-25 Yale University, Inc. Variant de kras et endométriose
US9150852B2 (en) 2011-02-18 2015-10-06 Raindance Technologies, Inc. Compositions and methods for molecular labeling
WO2012118745A1 (fr) 2011-02-28 2012-09-07 Arnold Oliphant Systèmes de dosage pour détection d'aneuploïdie et détermination du sexe
US8759036B2 (en) 2011-03-21 2014-06-24 Affymetrix, Inc. Methods for synthesizing pools of probes
US20140065615A1 (en) 2011-03-21 2014-03-06 Yale University The KRAS Variant and Tumor Biology
MX348655B (es) 2011-04-01 2017-06-21 Genentech Inc Biomarcadores para pronosticar sensibilidad a tratamientos contra el cancer.
GB201106254D0 (en) 2011-04-13 2011-05-25 Frisen Jonas Method and product
WO2012149193A2 (fr) 2011-04-29 2012-11-01 Monsanto Technology Llc Marqueurs moléculaires de diagnostic pour des caractères de pureté de lot de graines chez le soja
EP3560329A1 (fr) 2011-05-02 2019-10-30 Board of Regents of the University of Nebraska Plantes présentant des caractéristiques utiles et procédés associés
AU2012251027B2 (en) 2011-05-04 2015-03-26 Htg Molecular Diagnostics, Inc. Quantitative nuclease protection Assay (qNPA) and sequencing (qNPS) improvements
CN108342453A (zh) 2011-05-09 2018-07-31 富鲁达公司 基于探针的核酸检测
DK2710145T3 (en) * 2011-05-17 2016-02-22 Dxterity Diagnostics Inc METHOD AND COMPOSITIONS FOR THE DETECTION OF TARGET NUCLEIC ACIDS
SG10201605049QA (en) 2011-05-20 2016-07-28 Fluidigm Corp Nucleic acid encoding reactions
EP3709018A1 (fr) 2011-06-02 2020-09-16 Bio-Rad Laboratories, Inc. Appareil microfluidique pour l'identification de composants d'une reaction chimique
FI3461807T3 (fi) 2011-06-08 2023-09-07 Life Technologies Corp Uudenlaisten detergenttien suunnittelu ja kehitys pcr-järjestelmissä käyttöä varten
US9567628B2 (en) 2011-06-08 2017-02-14 Life Technologies Corporation Polymerization of nucleic acids using proteins having low isoelectric points
CA2838091C (fr) 2011-07-05 2019-08-13 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, Centers For Disease Control And Prevention Essai de genotypage de vih-1 pour la surveillance globale d'une resistance a un medicament de vih-1
WO2013010074A1 (fr) 2011-07-13 2013-01-17 Primeradx, Inc. Méthodes multimodales de détection et de quantification simultanées de plusieurs acides nucléiques dans un échantillon
US8658430B2 (en) 2011-07-20 2014-02-25 Raindance Technologies, Inc. Manipulating droplet size
US9670540B2 (en) 2011-07-21 2017-06-06 Cornell University Methods and devices for DNA sequencing and molecular diagnostics
PT3517615T (pt) 2011-08-31 2022-08-09 Seminis Vegetable Seeds Inc Métodos e composições para a firmeza da melancia
US8712697B2 (en) 2011-09-07 2014-04-29 Ariosa Diagnostics, Inc. Determination of copy number variations using binomial probability calculations
EP2761032B1 (fr) * 2011-09-29 2017-07-19 Luminex Corporation Sondes d'hydrolyse
US20140234360A1 (en) 2011-09-30 2014-08-21 The United States of America, as represented by the Secretary, Dept.of Health and Human Services Influenza vaccine
CA2851388C (fr) 2011-10-10 2023-11-21 The Hospital For Sick Children Methodes et compositions de depistage et de traitement de troubles du developpement
EP2769007B1 (fr) 2011-10-19 2016-12-07 Nugen Technologies, Inc. Compositions et procédés pour l'amplification et le séquençage directionnels d'acide nucléique
US11180807B2 (en) 2011-11-04 2021-11-23 Population Bio, Inc. Methods for detecting a genetic variation in attractin-like 1 (ATRNL1) gene in subject with Parkinson's disease
RU2014123511A (ru) 2011-11-10 2015-12-20 Дженентек, Инк Способы лечения, диагностики и мониторинга болезни альцгеймера
WO2013071954A1 (fr) 2011-11-15 2013-05-23 Université Libre de Bruxelles Détection de streptococcus pneumoniae dans le sang
SG11201402510TA (en) 2011-11-30 2014-06-27 Genentech Inc Erbb3 mutations in cancer
WO2013082308A1 (fr) 2011-11-30 2013-06-06 Children's Hospital Medical Center Gestion de la douleur et anesthésie personnalisées : identification préventive du risque et aide à la décision thérapeutique
US10648030B2 (en) 2012-01-13 2020-05-12 Affymetrix, Inc. Methods of determining the presence or absence of a plurality of target polynucleotides in a sample
WO2013112923A1 (fr) 2012-01-26 2013-08-01 Nugen Technologies, Inc. Compositions et procédés pour l'enrichissement en séquence d'acide nucléique ciblée et la génération d'une banque à efficacité élevée
WO2013120018A1 (fr) 2012-02-09 2013-08-15 Population Diagnostics, Inc. Méthodes et compositions permettant de rechercher et de traiter des troubles du développement
WO2013120089A1 (fr) * 2012-02-10 2013-08-15 Raindance Technologies, Inc. Dosage de type criblage diagnostique moléculaire
WO2013122826A1 (fr) * 2012-02-14 2013-08-22 Gnubio, Inc. Addition en cascade d'adaptateurs universels spécifiques cibles à des acides nucléiques
EP2820174B1 (fr) 2012-02-27 2019-12-25 The University of North Carolina at Chapel Hill Procédés et utilisations d'étiquettes moléculaires
ES2663234T3 (es) 2012-02-27 2018-04-11 Cellular Research, Inc Composiciones y kits para recuento molecular
CN104136611B (zh) * 2012-02-27 2018-03-27 东丽株式会社 核酸的检测方法
US9045803B2 (en) 2012-02-29 2015-06-02 Abbott Molecular Inc. Hepatitis B virus typing and resistance assay
AR090204A1 (es) 2012-02-29 2014-10-29 Dow Agrosciences Llc Potenciador viral baciliforme de caña de azucar (scbv) y su uso en la genomica funcional de plantas
US9428813B2 (en) 2012-03-26 2016-08-30 The United States Of America, As Represented By The Secretary, Dept. Of Health & Human Services DNA methylation analysis for the diagnosis, prognosis and treatment of adrenal neoplasms
US9803251B2 (en) 2012-05-03 2017-10-31 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Methods of detecting influenza virus
US10289800B2 (en) 2012-05-21 2019-05-14 Ariosa Diagnostics, Inc. Processes for calculating phased fetal genomic sequences
SG11201407901PA (en) 2012-05-21 2015-01-29 Fluidigm Corp Single-particle analysis of particle populations
AU2012380717B2 (en) 2012-05-24 2018-08-16 Fundacio Institucio Catalana De Recerca I Estudis Avancats Method for the identification of the origin of a cancer of unknown primary origin by methylation analysis
US9394574B2 (en) 2012-06-12 2016-07-19 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Methods for detecting Legionella nucleic acids in a sample
EP2861757B1 (fr) 2012-06-14 2019-11-06 Life Technologies Corporation Nouvelles compositions, nouveaux procédés et kits pour la réaction en chaîne de polymérase (pcr)
CA2877094A1 (fr) 2012-06-18 2013-12-27 Nugen Technologies, Inc. Compositions et procedes pour la selection negative de sequences d'acide nucleique indesirable
WO2013192292A1 (fr) 2012-06-21 2013-12-27 Justin Lamb Analyse de séquence d'acide nucléique spécifique d'un locus multiplexe massivement parallèle
US20150011396A1 (en) 2012-07-09 2015-01-08 Benjamin G. Schroeder Methods for creating directional bisulfite-converted nucleic acid libraries for next generation sequencing
CA2878280A1 (fr) 2012-07-19 2014-01-23 Ariosa Diagnostics, Inc. Detection a base de ligature sequentielle multiplexe de variants genetiques
US9951386B2 (en) 2014-06-26 2018-04-24 10X Genomics, Inc. Methods and systems for processing polynucleotides
US11591637B2 (en) 2012-08-14 2023-02-28 10X Genomics, Inc. Compositions and methods for sample processing
US10323279B2 (en) 2012-08-14 2019-06-18 10X Genomics, Inc. Methods and systems for processing polynucleotides
US9701998B2 (en) 2012-12-14 2017-07-11 10X Genomics, Inc. Methods and systems for processing polynucleotides
CA2881685C (fr) 2012-08-14 2023-12-05 10X Genomics, Inc. Compositions de microcapsule et procedes
US10273541B2 (en) 2012-08-14 2019-04-30 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10752949B2 (en) 2012-08-14 2020-08-25 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10584381B2 (en) 2012-08-14 2020-03-10 10X Genomics, Inc. Methods and systems for processing polynucleotides
DK2895621T3 (da) 2012-09-14 2020-11-30 Population Bio Inc Fremgangsmåder og sammensætning til diagnosticering, prognose og behandling af neurologiske tilstande
CA2922005A1 (fr) 2012-09-27 2014-04-03 Population Diagnostics, Inc. Procedes et compositions de depistage et de traitement de troubles du developpement
JP2015530113A (ja) 2012-09-28 2015-10-15 セファイド マイクロｒｎａ多重アッセイのための２プライマーｐｃｒ
EP3628746A1 (fr) 2012-11-02 2020-04-01 Life Technologies Corporation Capture, détection et quantification de petit arn
WO2014074942A1 (fr) 2012-11-08 2014-05-15 Illumina, Inc. Variants de risque de développer la maladie d'alzheimer
US10314253B2 (en) 2012-12-04 2019-06-11 Seminis Vegetable Seeds, Inc. Methods and compositions for watermelon sex expression
WO2014089536A1 (fr) * 2012-12-07 2014-06-12 Invitae Corporation Procédés de détection d'acide nucléique multiplexe
US10533221B2 (en) 2012-12-14 2020-01-14 10X Genomics, Inc. Methods and systems for processing polynucleotides
EP3567116A1 (fr) 2012-12-14 2019-11-13 10X Genomics, Inc. Procédés et systèmes de traitement de polynucléotides
AU2014205110A1 (en) * 2013-01-13 2015-08-27 Unitaq Bio Methods and compositions for PCR using blocked and universal primers
EP2954065B1 (fr) 2013-02-08 2021-07-28 10X Genomics, Inc. Fractionnement et traitement d'analytes et d'autres espèces
KR102182488B1 (ko) 2013-02-25 2020-11-24 제넨테크, 인크. 약물 내성 akt 돌연변이체의 검출 및 치료를 위한 방법 및 조성물
US9982255B2 (en) * 2013-03-11 2018-05-29 Kailos Genetics, Inc. Capture methodologies for circulating cell free DNA
US11254977B2 (en) 2013-03-12 2022-02-22 Life Technologies Corporation Universal reporter-based genotyping methods and materials
US10612088B2 (en) 2013-03-14 2020-04-07 The Broad Institute, Inc. Massively multiplexed RNA sequencing
US20140272959A1 (en) * 2013-03-14 2014-09-18 President And Fellows Of Harvard College Methods of Hybridizing Probes to Genomic DNA
US10450595B2 (en) 2013-03-15 2019-10-22 Theranos Ip Company, Llc Nucleic acid amplification
EP3564370B1 (fr) 2013-03-15 2022-01-12 Labrador Diagnostics LLC Amplification d'acides nucléiques
US10294489B2 (en) 2013-03-15 2019-05-21 Board Of Trustees Of Southern Illinois University Soybean resistant to cyst nematodes
WO2014145296A2 (fr) 2013-03-15 2014-09-18 Theranos, Inc. Amplification d'acide nucléique
CA2906076A1 (fr) 2013-03-15 2014-09-18 Abvitro, Inc. Attribution d'un code-barres a des cellules isolees pour la decouverte d'anticorps
EP2970961B1 (fr) 2013-03-15 2019-04-24 Theranos IP Company, LLC Amplification d'acide nucléique
WO2014144092A1 (fr) 2013-03-15 2014-09-18 Nugen Technologies, Inc. Séquençage séquentiel
US9822418B2 (en) 2013-04-22 2017-11-21 Icahn School Of Medicine At Mount Sinai Mutations in PDGFRB and NOTCH3 as causes of autosomal dominant infantile myofibromatosis
US10059999B2 (en) 2013-06-10 2018-08-28 Monsanto Technology Llc Molecular markers associated with soybean tolerance to low iron growth conditions
WO2014200579A1 (fr) 2013-06-13 2014-12-18 Ariosa Diagnostics, Inc. Analyse statistique pour détermination non-invasive d'une l'aneuploïdie des chromosomes sexuels
WO2014210225A1 (fr) 2013-06-25 2014-12-31 Prognosys Biosciences, Inc. Procédés et systèmes pour déterminer des motifs spatiales de cibles biologiques dans un échantillon
SG11201601201VA (en) * 2013-08-19 2016-03-30 Singular Bio Inc Assays for single molecule detection and use thereof
EP3039158B1 (fr) 2013-08-28 2018-11-14 Cellular Research, Inc. Analyse massivement parallèle de cellules uniques
EP3041957A4 (fr) 2013-09-04 2017-03-29 Fluidigm Corporation Dosages de proximité pour détecter des acides nucléiques et des protéines dans une cellule unique
US10767188B2 (en) 2013-09-25 2020-09-08 Nutech Ventures Methods and compositions for obtaining useful plant traits
US11901041B2 (en) 2013-10-04 2024-02-13 Bio-Rad Laboratories, Inc. Digital analysis of nucleic acid modification
JP2017504307A (ja) 2013-10-07 2017-02-09 セルラー リサーチ， インコーポレイテッド アレイ上のフィーチャーをデジタルカウントするための方法およびシステム
EP3060686B1 (fr) 2013-10-22 2018-08-01 The Government of the United States of America as represented by the Secretary of the Department of Health and Human Services Compositions et procédés permettant de détecter des virus de la grippe
EP3539944A1 (fr) 2013-10-25 2019-09-18 Life Technologies Corporation Nouveaux composés à utiliser dans des systèmes acp et applications correspondantes
EP2891722B1 (fr) 2013-11-12 2018-10-10 Population Bio, Inc. Procédés et compositions de diagnostic, pronostic et traitement de l'endométriose
US10364465B2 (en) 2013-11-12 2019-07-30 Life Technologies Corporation Reagents and methods for sequencing
EP3068883B1 (fr) 2013-11-13 2020-04-29 Nugen Technologies, Inc. Compositions et procédés pour l'identification d'une lecture de séquençage en double
CN105765080A (zh) * 2013-11-26 2016-07-13 伯乐实验室公司 用于检测核酸相邻性的方法
NZ784204A (en) 2013-11-27 2022-10-28 Seminis Vegetable Seeds Inc Disease resistance loci in onion
US9944977B2 (en) 2013-12-12 2018-04-17 Raindance Technologies, Inc. Distinguishing rare variations in a nucleic acid sequence from a sample
US9824068B2 (en) 2013-12-16 2017-11-21 10X Genomics, Inc. Methods and apparatus for sorting data
EP2986742A4 (fr) * 2014-01-10 2016-12-07 Bio Rad Laboratories Inc Colorants intercalants de détection différentielle
US10260111B1 (en) 2014-01-20 2019-04-16 Brett Eric Etchebarne Method of detecting sepsis-related microorganisms and detecting antibiotic-resistant sepsis-related microorganisms in a fluid sample
WO2015127201A1 (fr) 2014-02-21 2015-08-27 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Acides nucléiques du vih-2 et leurs procédés de détection
CA3080638C (fr) 2014-02-21 2023-07-11 Syngenta Participations Ag Loci genetiques associes a la fertilite accrue dans le mais
EP3110968B1 (fr) 2014-02-24 2019-04-17 Children's Hospital Medical Center Méthodes et compositions pour une gestion personnalisée de la douleur
NZ630710A (en) 2014-02-27 2016-03-31 Seminis Vegetable Seeds Inc Compositions and methods for peronospora resistance in spinach
WO2015131107A1 (fr) 2014-02-28 2015-09-03 Nugen Technologies, Inc. Séquençage au bisulfite de représentation réduite avec adaptateurs de diversité
WO2015147370A1 (fr) * 2014-03-28 2015-10-01 Seegene, Inc. Détection de séquences d'acide nucléique cible à l'aide de différentes températures de détection
WO2015153732A2 (fr) 2014-04-01 2015-10-08 Cornell University Utilisation d'adn double brin dans des exosomes : un nouveau biomarqueur dans la détection du cancer
ES2807222T3 (es) 2014-04-01 2021-02-22 Univ Cornell Detección de metilación de ADN usando reacciones combinadas de ligamiento y nucleasa
EP3126524B1 (fr) * 2014-04-04 2020-07-22 Affymetrix, Inc. Compositions et procédés améliorés pour analyses au moyen de sondes d'inversion moléculaire
AU2015243445B2 (en) 2014-04-10 2020-05-28 10X Genomics, Inc. Fluidic devices, systems, and methods for encapsulating and partitioning reagents, and applications of same
WO2015160536A1 (fr) 2014-04-14 2015-10-22 The United States Of America, As Represented By The Secretary Department Of Health And Human Services Procédés de détection et d'identification rapides d'acides nucléiques viraux
CN107075509B (zh) 2014-05-23 2021-03-09 数字基因公司 通过数字化转座子的单倍体组测定
CN113046413A (zh) 2014-06-06 2021-06-29 康奈尔大学 从血液特异性靶向捕获人类基因组和转录组区域的方法
WO2015199976A1 (fr) 2014-06-24 2015-12-30 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Microdissection activée par une cible
CN106795553B (zh) 2014-06-26 2021-06-04 10X基因组学有限公司 分析来自单个细胞或细胞群体的核酸的方法
US10316369B2 (en) 2014-06-27 2019-06-11 Seminis Vegetable Seeds, Inc. Methods and assays for male sterile watermelon
EP3175001B1 (fr) 2014-07-30 2021-02-10 Mor Research Applications Ltd. Méthodes de pronostic et systèmes de traitement de la leucémie lymphoblastique aiguë
GB201413718D0 (en) * 2014-08-01 2014-09-17 Olink Ab Method for selecting a target nucleic acid sequence
EP3177738B1 (fr) 2014-08-08 2019-10-09 Children's Hospital Medical Center Procédé de diagnostic permettant de distinguer des formes de l'éosinophilie oesophagienne
CN108064315B (zh) 2014-08-22 2022-01-18 西菲伊德公司 检测流感的方法
GB2558326B (en) 2014-09-05 2021-01-20 Population Bio Inc Methods and compositions for inhibiting and treating neurological conditions
US9856521B2 (en) 2015-01-27 2018-01-02 BioSpyder Technologies, Inc. Ligation assays in liquid phase
US11091810B2 (en) 2015-01-27 2021-08-17 BioSpyder Technologies, Inc. Focal gene expression profiling of stained FFPE tissues with spatial correlation to morphology
US10683534B2 (en) 2015-01-27 2020-06-16 BioSpyder Technologies, Inc. Ligation assays in liquid phase
EP3950944A1 (fr) 2014-09-15 2022-02-09 AbVitro LLC Séquençage à haut débit de banque de nucléotides
CA2961209A1 (fr) * 2014-09-17 2016-03-24 Theranos, Inc. Procedes et compositions de diagnostic
EP3005862A1 (fr) 2014-10-10 2016-04-13 Seminis Vegetable Seeds, Inc. Plants de melon présentant une meilleure tolérance aux maladies
WO2016061111A1 (fr) 2014-10-13 2016-04-21 Life Technologies Corporation Procédés, kits et compositions pour déterminer des nombres de copies de gène
WO2016069853A2 (fr) 2014-10-30 2016-05-06 Cepheid Méthodes de détection d'ebola
US9975122B2 (en) 2014-11-05 2018-05-22 10X Genomics, Inc. Instrument systems for integrated sample processing
JP6629224B2 (ja) * 2014-11-06 2020-01-15 公立大学法人大阪 クランピングプローブ
US11279974B2 (en) 2014-12-01 2022-03-22 The Broad Institute, Inc. Method for in situ determination of nucleic acid proximity
US10085951B2 (en) 2014-12-11 2018-10-02 Designs For Health, Inc. Curcuminoid formulations and related methods of treatment
WO2016100388A1 (fr) 2014-12-15 2016-06-23 Cepheid Amplification d'acides nucléiques supérieure à 2 sur une base exponentielle
CN107109398B (zh) * 2014-12-19 2020-08-18 荣研化学株式会社 单核苷酸多态性检测用寡核苷酸探针及单核苷酸多态性检测方法
KR101728023B1 (ko) * 2015-01-02 2017-04-18 주식회사 랩 지노믹스 Ｐｃｒ―ｌｄｒ을 이용한 ａｔｐ７ｂ 유전자의 돌연변이 검출
CN112126675B (zh) 2015-01-12 2022-09-09 10X基因组学有限公司 用于制备核酸测序文库的方法和系统以及用其制备的文库
US9984201B2 (en) 2015-01-18 2018-05-29 Youhealth Biotech, Limited Method and system for determining cancer status
WO2016123419A1 (fr) * 2015-01-30 2016-08-04 President And Fellows Of Harvard College Imagerie sans microscope
US10697010B2 (en) 2015-02-19 2020-06-30 Becton, Dickinson And Company High-throughput single-cell analysis combining proteomic and genomic information
WO2016137973A1 (fr) 2015-02-24 2016-09-01 10X Genomics Inc Procédés et systèmes de traitement de cloisonnement
US20160246813A1 (en) * 2015-02-25 2016-08-25 International Business Machines Corporation System and method for machine information life cycle
EP3262214B1 (fr) 2015-02-27 2023-10-25 Standard BioTools Inc. Dispositif microfluidique
WO2016138496A1 (fr) 2015-02-27 2016-09-01 Cellular Research, Inc. Codage à barres moléculaire à adressage spatial
WO2016145409A1 (fr) 2015-03-11 2016-09-15 The Broad Institute, Inc. Couplage de génotype et de phénotype
US9434996B1 (en) 2015-03-13 2016-09-06 Tracy Ann Hayden All mini-STR multiplex with increased C.E. through-put by STR prolongation template fusion
WO2016160844A2 (fr) 2015-03-30 2016-10-06 Cellular Research, Inc. Procédés et compositions pour codage à barres combinatoire
US20160299129A1 (en) * 2015-04-07 2016-10-13 Xiaolei Qiu Ultra Sensitive and Specific Multiplex Biosensor System Based on Multiple Cooperative Interactions
US10774374B2 (en) 2015-04-10 2020-09-15 Spatial Transcriptomics AB and Illumina, Inc. Spatially distinguished, multiplex nucleic acid analysis of biological specimens
WO2016170121A1 (fr) * 2015-04-23 2016-10-27 Pathofinder B.V. Procédé de détection simultanée de multiples séquences d'acide nucléique dans un échantillon
WO2016172373A1 (fr) * 2015-04-23 2016-10-27 Cellular Research, Inc. Procédés et compositions pour l'amplification de transcriptome entier
MX2017013872A (es) 2015-04-28 2018-03-12 Monsanto Technology Llc Metodos y composiciones para producir plantas de maiz braquiticas.
UA126326C2 (uk) 2015-04-30 2022-09-21 Монсанто Текнолоджі Елелсі Спосіб відбору рослини каноли, стійкої до кили капустяних
US11124823B2 (en) 2015-06-01 2021-09-21 Becton, Dickinson And Company Methods for RNA quantification
ES2842205T3 (es) 2015-06-15 2021-07-13 Cepheid Integración de la purificación del ADN y la medición de su metilación y la medición conjunta de mutaciones y/o niveles de expresión de ARNm en un cartucho de reacción automático
JP6830094B2 (ja) 2015-07-29 2021-02-17 プロジェニティ， インコーポレイテッド 染色体異常を検出するための核酸及び方法
WO2017031370A1 (fr) 2015-08-18 2017-02-23 The Broad Institute, Inc. Procédés et compositions permettant de changer la fonction et la structure de boucles et/ou de domaines de chromatine
CR20180164A (es) 2015-08-18 2018-09-04 Monsanto Technology Llc Métodos para producir plantas de algodón con tolerancia mejorada a la sequía y composiciones de estas
US10448595B2 (en) 2015-09-03 2019-10-22 Seminis Vegetable Seeds, Inc. Downy mildew resistant lettuce plants
US11118216B2 (en) 2015-09-08 2021-09-14 Affymetrix, Inc. Nucleic acid analysis by joining barcoded polynucleotide probes
US10858709B2 (en) 2015-09-10 2020-12-08 Monsanto Technology Llc Methods for producing corn plants with downy mildew resistance and compositions thereof
ES2745694T3 (es) 2015-09-11 2020-03-03 Cellular Res Inc Métodos y composiciones para normalización de biblioteca de ácido nucleico
EP3353325B1 (fr) 2015-09-24 2024-03-20 AbVitro LLC Acp par exclusion activée à produit d'amplification unique
CA2999888A1 (fr) 2015-09-24 2017-03-30 Abvitro Llc Conjuges affinite-oligonucleotide et leurs utilisations
US10928392B2 (en) 2015-09-25 2021-02-23 Abvitro Llc High throughput process for T cell receptor target identification of natively-paired T cell receptor sequences
EP3371211A4 (fr) 2015-11-04 2019-08-21 Icahn School of Medicine at Mount Sinai Méthodes de traitement de tumeurs et du cancer, et identification de sujets candidats à ce traitement
EP3377647B1 (fr) 2015-11-16 2020-03-11 Progenity, Inc. Acides nucléiques et procédés de détection de l'état de méthylation
US11371094B2 (en) 2015-11-19 2022-06-28 10X Genomics, Inc. Systems and methods for nucleic acid processing using degenerate nucleotides
SG11201803983UA (en) * 2015-11-19 2018-06-28 10X Genomics Inc Transformable tagging compositions, methods, and processes incorporating same
JP6703824B2 (ja) * 2015-11-30 2020-06-03 シスメックス株式会社 細胞選択方法、細胞検出方法、細胞選択装置、および細胞検出装置
US10774370B2 (en) 2015-12-04 2020-09-15 10X Genomics, Inc. Methods and compositions for nucleic acid analysis
EP3390658B1 (fr) 2015-12-16 2022-08-03 Standard BioTools Inc. Amplification multiplex de haut niveau
US10694693B2 (en) 2015-12-18 2020-06-30 Monsanto Technology Llc Methods for producing corn plants with northern leaf blight resistance and compositions thereof
AU2017207341A1 (en) 2016-01-12 2018-08-02 Interleukin Genetics, Inc. Methods for predicting response to treatment
EP3402572B1 (fr) 2016-01-13 2022-03-16 Children's Hospital Medical Center Compositions et méthodes de traitement d'états inflammatoires allergiques
SG11201806757XA (en) 2016-02-11 2018-09-27 10X Genomics Inc Systems, methods, and media for de novo assembly of whole genome sequence data
US11246868B2 (en) 2016-04-26 2022-02-15 Icahn School Of Medicine At Mount Sinai Treatment of hippo pathway mutant tumors and methods of identifying subjects as candidates for treatment
EP3449015A1 (fr) 2016-04-27 2019-03-06 Mira DX, Inc. Traitement à base immunitaire de patients atteints du cancer à variantkras
ES2956757T3 (es) 2016-05-02 2023-12-27 Becton Dickinson Co Codificación con códigos de barras moleculares precisa
WO2017197338A1 (fr) 2016-05-13 2017-11-16 10X Genomics, Inc. Systèmes microfluidiques et procédés d'utilisation
US10301677B2 (en) 2016-05-25 2019-05-28 Cellular Research, Inc. Normalization of nucleic acid libraries
US11397882B2 (en) 2016-05-26 2022-07-26 Becton, Dickinson And Company Molecular label counting adjustment methods
US10640763B2 (en) 2016-05-31 2020-05-05 Cellular Research, Inc. Molecular indexing of internal sequences
US10202641B2 (en) 2016-05-31 2019-02-12 Cellular Research, Inc. Error correction in amplification of samples
EP3464642A4 (fr) 2016-05-31 2020-02-19 The Regents of the University of California Procédés d'évaluation, de surveillance et de modulation du processus de vieillissement
EP3472349A1 (fr) 2016-06-16 2019-04-24 Life Technologies Corporation Nouvelles compositions, procédés et kits permettant de détecter des micro-organismes
WO2018005284A1 (fr) 2016-06-27 2018-01-04 The United State Of America, As Represented By The Secretary, Department Of Health And Human Services Procédés et compositions de sous-typage du virus de la grippe a
US10093986B2 (en) 2016-07-06 2018-10-09 Youhealth Biotech, Limited Leukemia methylation markers and uses thereof
US11396678B2 (en) 2016-07-06 2022-07-26 The Regent Of The University Of California Breast and ovarian cancer methylation markers and uses thereof
WO2018009707A1 (fr) 2016-07-06 2018-01-11 Youhealth Biotech, Limited Marqueurs de méthylation spécifiques d'une tumeur solide et utilisations de ces marqueurs
US10822648B1 (en) 2016-07-29 2020-11-03 Labrador Diagnostics Llc Hybrid multi-step nucleic acid amplification
EP3496528A4 (fr) 2016-08-11 2020-03-18 Monsanto Technology LLC Procédés et compositions permettant de produire des plants de maïs dotés d'une résistance au flétrissement tardif
WO2018039599A1 (fr) 2016-08-26 2018-03-01 Life Technologies Corporation Commandes d'extraction et d'amplification d'acides nucléiques et procédés d'utilisation associés
WO2018057502A2 (fr) 2016-09-20 2018-03-29 President And Fellows Harvard College Systèmes de vérification moléculaire
JP6929354B2 (ja) 2016-09-24 2021-09-01 アブビトロ， エルエルシー 親和性−オリゴヌクレオチドコンジュゲートおよびその使用
KR102363716B1 (ko) 2016-09-26 2022-02-18 셀룰러 리서치, 인크. 바코딩된 올리고뉴클레오티드 서열을 갖는 시약을 이용한 단백질 발현의 측정
AU2017232187B2 (en) 2016-09-30 2023-11-09 Seminis Vegetable Seeds, Inc. Xanthomonas resistant brassica oleracea plants
US11725232B2 (en) 2016-10-31 2023-08-15 The Hong Kong University Of Science And Technology Compositions, methods and kits for detection of genetic variants for alzheimer's disease
UA127452C2 (uk) 2016-12-06 2023-08-30 Орегон Стейт Юніверсіті Бактерія streptomyces fungicidicus та спосіб одержання ендурацидину в штамі streptomyces fungicidicus
BR112019011670A2 (pt) 2016-12-12 2019-10-22 Cepheid purificação integrada e mensuração da metilação do dna e comensuração de mutações e/ou níveis de expressão de mrna em um cartucho de reação automático
WO2018111782A1 (fr) 2016-12-12 2018-06-21 Cepheid Immuno-pcr et analyse d'acide nucléique co-intégrées dans une cartouche de réaction automatisée
WO2018118808A1 (fr) 2016-12-19 2018-06-28 The Broad Institute, Inc. Méthodes de traitement des troubles du spectre autistique
US10815525B2 (en) 2016-12-22 2020-10-27 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10550429B2 (en) 2016-12-22 2020-02-04 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10337070B2 (en) 2017-01-12 2019-07-02 Cardioforecast Ltd. Methods and kits for treating cardiovascular disease
EP3568234B1 (fr) 2017-01-13 2023-09-06 Cellular Research, Inc. Revêtement hydrophile des canaux fluidiques
US11618924B2 (en) 2017-01-20 2023-04-04 Children's Hospital Medical Center Methods and compositions relating to OPRM1 DNA methylation for personalized pain management
WO2018140966A1 (fr) 2017-01-30 2018-08-02 10X Genomics, Inc. Procédés et systèmes de codage à barres de cellules individuelles sur la base de gouttelettes
US11319583B2 (en) 2017-02-01 2022-05-03 Becton, Dickinson And Company Selective amplification using blocking oligonucleotides
US10240205B2 (en) 2017-02-03 2019-03-26 Population Bio, Inc. Methods for assessing risk of developing a viral disease using a genetic test
US10995333B2 (en) 2017-02-06 2021-05-04 10X Genomics, Inc. Systems and methods for nucleic acid preparation
BR112019018272A2 (pt) 2017-03-02 2020-07-28 Youhealth Oncotech, Limited marcadores metilação para diagnosticar hepatocelular carcinoma e câncer
WO2018175399A1 (fr) 2017-03-24 2018-09-27 Bio-Rad Laboratories, Inc. Amorces universelles en épingle à cheveux
KR20190140952A (ko) 2017-04-24 2019-12-20 제넨테크, 인크. 막관통 및 인접막 도메인에서의 erbb2/her2 돌연변이
US11708613B2 (en) 2017-05-03 2023-07-25 The United States of America, as Represened by the Secretary, Department of Health and Human Services Rapid detection of Zika virus by reverse transcription loop-mediated isothermal amplification
WO2018213803A1 (fr) 2017-05-19 2018-11-22 Neon Therapeutics, Inc. Identification de néo-antigène immunogène
WO2018218222A1 (fr) 2017-05-26 2018-11-29 Goldfless Stephen Jacob Séquençage de bibliothèque de polynucléotides à haut rendement et analyse de transcriptome
EP4230746A3 (fr) 2017-05-26 2023-11-01 10X Genomics, Inc. Analyse de cellule unique de chromatine accessible par transposase
US10844372B2 (en) 2017-05-26 2020-11-24 10X Genomics, Inc. Single cell analysis of transposase accessible chromatin
JP2020522262A (ja) 2017-06-05 2020-07-30 ベクトン・ディキンソン・アンド・カンパニーＢｅｃｔｏｎ， Ｄｉｃｋｉｎｓｏｎ Ａｎｄ Ｃｏｍｐａｎｙ 単一細胞用のサンプルインデックス付加
WO2018232028A1 (fr) 2017-06-14 2018-12-20 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Détection de gènes de résistance antibactérienne blaimp
EP3679370A1 (fr) 2017-09-07 2020-07-15 Juno Therapeutics, Inc. Procédés d'identification de caractéristiques cellulaires relatives à des réponses associées à une thérapie cellulaire
WO2019055829A1 (fr) 2017-09-15 2019-03-21 Nazarian Javad Procédés de détection de biomarqueurs du cancer
ES2924185T3 (es) 2017-09-25 2022-10-05 Fred Hutchinson Cancer Center Perfiles in situ de alta eficiencia dirigidos a todo el genoma
US10837047B2 (en) 2017-10-04 2020-11-17 10X Genomics, Inc. Compositions, methods, and systems for bead formation using improved polymers
US11099202B2 (en) 2017-10-20 2021-08-24 Tecan Genomics, Inc. Reagent delivery system
WO2019084043A1 (fr) 2017-10-26 2019-05-02 10X Genomics, Inc. Méthodes et systèmes de préparation d'acide nucléique et d'analyse de chromatine
EP4241882A3 (fr) 2017-10-27 2023-12-06 10X Genomics, Inc. Procédés de préparation et d'analyse d'échantillons
WO2019094973A1 (fr) 2017-11-13 2019-05-16 Life Technologies Corporation Compositions, procédés et kits de détection de micro-organismes dans les voies urinaires
EP3954782A1 (fr) 2017-11-15 2022-02-16 10X Genomics, Inc. Perles de gel fonctionnalisées
US10829815B2 (en) 2017-11-17 2020-11-10 10X Genomics, Inc. Methods and systems for associating physical and genetic properties of biological particles
WO2019108851A1 (fr) 2017-11-30 2019-06-06 10X Genomics, Inc. Systèmes et procédés de préparation et d'analyse d'acides nucléiques
EP3728636A1 (fr) 2017-12-19 2020-10-28 Becton, Dickinson and Company Particules associées à des oligonucléotides
WO2019133727A1 (fr) 2017-12-29 2019-07-04 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Ensemble de sondes universelles de virus de la grippe pour l'enrichissement d'un quelconque acide nucléique du virus de la grippe
US11814674B2 (en) 2018-02-02 2023-11-14 The United States Of America, As Represented By The Secretary Department Of Health And Human Services Random amplification methods for extremely low input nucleic acids
WO2019157529A1 (fr) 2018-02-12 2019-08-15 10X Genomics, Inc. Procédés de caractérisation d'analytes multiples à partir de cellules individuelles ou de populations cellulaires
US11639928B2 (en) 2018-02-22 2023-05-02 10X Genomics, Inc. Methods and systems for characterizing analytes from individual cells or cell populations
US11859250B1 (en) 2018-02-23 2024-01-02 Children's Hospital Medical Center Methods for treating eosinophilic esophagitis
AU2019247652A1 (en) 2018-04-02 2020-10-15 Enumera Molecular, Inc. Methods, systems, and compositions for counting nucleic acid molecules
CN112262218A (zh) 2018-04-06 2021-01-22 10X基因组学有限公司 用于单细胞处理中的质量控制的系统和方法
US20190323066A1 (en) * 2018-04-20 2019-10-24 Longhorn Vaccines And Diagnostics, Llc Rapid Methods for the Detection of Microbial Resistance
CN112243461A (zh) 2018-05-03 2021-01-19 贝克顿迪金森公司 在相对的转录物末端进行分子条形码化
EP4234717A3 (fr) 2018-05-03 2023-11-01 Becton, Dickinson and Company Analyse multi-omique d'échantillons à haut débit
WO2019213619A1 (fr) 2018-05-04 2019-11-07 Abbott Laboratories Méthodes et produits de diagnostic, de pronostic et de thérapie du vhb
CN108531599A (zh) * 2018-05-08 2018-09-14 杭州泰领生物技术有限公司 一种人hla-b*5801基因型的试剂盒及方法
US11268102B2 (en) 2018-05-16 2022-03-08 University Of Florida Research Foundation, Incorporated Compositions and methods for identifying and selecting brachytic locus in solanaceae
US11932899B2 (en) 2018-06-07 2024-03-19 10X Genomics, Inc. Methods and systems for characterizing nucleic acid molecules
US11703427B2 (en) 2018-06-25 2023-07-18 10X Genomics, Inc. Methods and systems for cell and bead processing
US20200032335A1 (en) 2018-07-27 2020-01-30 10X Genomics, Inc. Systems and methods for metabolome analysis
JP2021532152A (ja) 2018-07-27 2021-11-25 アペルタ バイオサイエンシズ，エルエルシー デモデックスにより誘発される眼の異常および顔面の異常を治療するためのスピノシン配合
SI3625368T1 (sl) 2018-08-08 2023-04-28 Pml Screening, Llc Postopki z genetskim testiranjem za oceno tveganja za razvoj progresivne multifokalne levkoencefalopatije, ki jo povzroča virus John Cunningham
US11519033B2 (en) 2018-08-28 2022-12-06 10X Genomics, Inc. Method for transposase-mediated spatial tagging and analyzing genomic DNA in a biological sample
CN112805389A (zh) 2018-10-01 2021-05-14 贝克顿迪金森公司 确定5’转录物序列
CN113227398A (zh) 2018-10-29 2021-08-06 塞弗德公司 在减少的扩增时间下使用巢式重叠引物的指数基数-3核酸扩增
WO2020097315A1 (fr) 2018-11-08 2020-05-14 Cellular Research, Inc. Analyse transcriptomique complète de cellules uniques à l'aide d'un amorçage aléatoire
US11459607B1 (en) 2018-12-10 2022-10-04 10X Genomics, Inc. Systems and methods for processing-nucleic acid molecules from a single cell using sequential co-partitioning and composite barcodes
WO2020123316A2 (fr) 2018-12-10 2020-06-18 10X Genomics, Inc. Procédés de détermination d'un emplacement d'un analyte biologique dans un échantillon biologique
US11492660B2 (en) 2018-12-13 2022-11-08 Becton, Dickinson And Company Selective extension in single cell whole transcriptome analysis
US11926867B2 (en) 2019-01-06 2024-03-12 10X Genomics, Inc. Generating capture probes for spatial analysis
US11649485B2 (en) 2019-01-06 2023-05-16 10X Genomics, Inc. Generating capture probes for spatial analysis
US11845983B1 (en) 2019-01-09 2023-12-19 10X Genomics, Inc. Methods and systems for multiplexing of droplet based assays
WO2020150144A1 (fr) 2019-01-15 2020-07-23 Seminis Vegetable Seeds, Inc. Plantes de haricot vert présentant une résistance améliorée aux maladies
US11371076B2 (en) 2019-01-16 2022-06-28 Becton, Dickinson And Company Polymerase chain reaction normalization through primer titration
EP3914728B1 (fr) 2019-01-23 2023-04-05 Becton, Dickinson and Company Oligonucléotides associés à des anticorps
SG11202108788TA (en) 2019-02-12 2021-09-29 10X Genomics Inc Methods for processing nucleic acid molecules
US11467153B2 (en) 2019-02-12 2022-10-11 10X Genomics, Inc. Methods for processing nucleic acid molecules
US11851683B1 (en) 2019-02-12 2023-12-26 10X Genomics, Inc. Methods and systems for selective analysis of cellular samples
US11655499B1 (en) 2019-02-25 2023-05-23 10X Genomics, Inc. Detection of sequence elements in nucleic acid molecules
US11920183B2 (en) 2019-03-11 2024-03-05 10X Genomics, Inc. Systems and methods for processing optically tagged beads
US11578373B2 (en) 2019-03-26 2023-02-14 Dermtech, Inc. Gene classifiers and uses thereof in skin cancers
WO2020206170A1 (fr) 2019-04-02 2020-10-08 Progenity, Inc. Procédés, systèmes et compositions de comptage de molécules d'acide nucléique
US20220249660A1 (en) 2019-06-06 2022-08-11 Sitokine Limited Compositions and methods for treating lung, colorectal and breast cancer
EP4004231A1 (fr) 2019-07-22 2022-06-01 Becton, Dickinson and Company Dosage de séquençage par immunoprécipitation de la chromatine monocellulaire
WO2021028469A1 (fr) 2019-08-12 2021-02-18 Sitokine Limited Compositions et méthodes de traitement du syndrome de libération de cytokines et de neurotoxicité
US11287422B2 (en) 2019-09-23 2022-03-29 Element Biosciences, Inc. Multivalent binding composition for nucleic acid analysis
SG10202008262UA (en) 2019-09-26 2021-04-29 Seminis Vegetable Seeds Inc Lettuce plants having resistance to nasonovia ribisnigri biotype nr:1
US11844800B2 (en) 2019-10-30 2023-12-19 Massachusetts Institute Of Technology Methods and compositions for predicting and preventing relapse of acute lymphoblastic leukemia
EP3901286A1 (fr) 2020-04-24 2021-10-27 Mirnax Biosens, S.L. Amorce inverse bivalente
US20220389497A1 (en) 2019-11-04 2022-12-08 Mirnax Biosens, S.L. Bivalent reverse primer
US11773436B2 (en) 2019-11-08 2023-10-03 Becton, Dickinson And Company Using random priming to obtain full-length V(D)J information for immune repertoire sequencing
WO2021092433A2 (fr) 2019-11-08 2021-05-14 10X Genomics, Inc. Amélioration de la spécificité de la liaison d'un analyte
EP4055185A1 (fr) 2019-11-08 2022-09-14 10X Genomics, Inc. Agents de capture d'analytes marqués spatialement pour le multiplexage d'analytes
FI3891300T3 (fi) 2019-12-23 2023-05-10 10X Genomics Inc Menetelmät spatiaalista analyysiä varten rna-templatoitua ligaatiota käyttäen
WO2021146207A1 (fr) 2020-01-13 2021-07-22 Becton, Dickinson And Company Procédés et compositions pour la quantification de protéines et d'arn
US11732299B2 (en) 2020-01-21 2023-08-22 10X Genomics, Inc. Spatial assays with perturbed cells
US11702693B2 (en) 2020-01-21 2023-07-18 10X Genomics, Inc. Methods for printing cells and generating arrays of barcoded cells
US11821035B1 (en) 2020-01-29 2023-11-21 10X Genomics, Inc. Compositions and methods of making gene expression libraries
US11898205B2 (en) 2020-02-03 2024-02-13 10X Genomics, Inc. Increasing capture efficiency of spatial assays
US11732300B2 (en) 2020-02-05 2023-08-22 10X Genomics, Inc. Increasing efficiency of spatial analysis in a biological sample
US11835462B2 (en) 2020-02-11 2023-12-05 10X Genomics, Inc. Methods and compositions for partitioning a biological sample
US11891654B2 (en) 2020-02-24 2024-02-06 10X Genomics, Inc. Methods of making gene expression libraries
US11926863B1 (en) 2020-02-27 2024-03-12 10X Genomics, Inc. Solid state single cell method for analyzing fixed biological cells
US11768175B1 (en) 2020-03-04 2023-09-26 10X Genomics, Inc. Electrophoretic methods for spatial analysis
WO2021205013A1 (fr) 2020-04-09 2021-10-14 Sitokine Limited Compositions et méthodes de traitement de la covid-19
EP4242325A3 (fr) 2020-04-22 2023-10-04 10X Genomics, Inc. Procédés d'analyse spatiale utilisant un appauvrissement d'arn ciblée
US11851700B1 (en) 2020-05-13 2023-12-26 10X Genomics, Inc. Methods, kits, and compositions for processing extracellular molecules
CN115605614A (zh) 2020-05-14 2023-01-13 贝克顿迪金森公司（Us） 用于免疫组库谱分析的引物
EP4153776A1 (fr) 2020-05-22 2023-03-29 10X Genomics, Inc. Analyse spatiale pour détecter des variants de séquence
EP4153775A1 (fr) 2020-05-22 2023-03-29 10X Genomics, Inc. Mesure spatio-temporelle simultanée de l'expression génique et de l'activité cellulaire
WO2021242834A1 (fr) 2020-05-26 2021-12-02 10X Genomics, Inc. Procédé de réinitialisation d'un réseau
AU2021283184A1 (en) 2020-06-02 2023-01-05 10X Genomics, Inc. Spatial transcriptomics for antigen-receptors
EP4025692A2 (fr) 2020-06-02 2022-07-13 10X Genomics, Inc. Procédés de banques d'acides nucléiques
WO2021252499A1 (fr) 2020-06-08 2021-12-16 10X Genomics, Inc. Méthodes de détermination de marge chirurgicale et méthodes d'utilisation associées
WO2021252591A1 (fr) 2020-06-10 2021-12-16 10X Genomics, Inc. Procédés de détermination d'un emplacement d'un analyte dans un échantillon biologique
AU2021294334A1 (en) 2020-06-25 2023-02-02 10X Genomics, Inc. Spatial analysis of DNA methylation
EP4172363A1 (fr) 2020-06-26 2023-05-03 Cepheid Procédés de détection du sras-cov-2, de la grippe et du rsv
US11761038B1 (en) 2020-07-06 2023-09-19 10X Genomics, Inc. Methods for identifying a location of an RNA in a biological sample
US11932901B2 (en) 2020-07-13 2024-03-19 Becton, Dickinson And Company Target enrichment using nucleic acid probes for scRNAseq
AU2021204717A1 (en) 2020-07-15 2022-02-03 Seminis Vegetable Seeds, Inc. Green Bean Plants with Improved Disease Resistance
CA3186950A1 (fr) 2020-07-23 2022-01-27 Scott Benson Compositions, systemes et methodes d'analyse biologique impliquant des conjugues de colorants a transfert d'energie et analytes comprenant ceux-ci
WO2022020723A1 (fr) 2020-07-23 2022-01-27 Life Technologies Corporation Conjugués de colorant de transfert d'énergie destinés à être utilisés dans des dosages biologiques
WO2022056078A1 (fr) 2020-09-11 2022-03-17 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Dosage de détection assisté par rnase h d'arn (radar)
EP4214243A1 (fr) 2020-09-21 2023-07-26 Progenity, Inc. Compositions et procédés d'isolement d'adn acellulaire
US11926822B1 (en) 2020-09-23 2024-03-12 10X Genomics, Inc. Three-dimensional spatial analysis
US20220106627A1 (en) 2020-10-06 2022-04-07 Cepheid Methods of diagnosing tuberculosis and differentiating between active and latent tuberculosis
US11827935B1 (en) 2020-11-19 2023-11-28 10X Genomics, Inc. Methods for spatial analysis using rolling circle amplification and detection probes
EP4247967A1 (fr) 2020-11-20 2023-09-27 Becton, Dickinson and Company Profilage de protéines hautement exprimées et faiblement exprimées
WO2022140028A1 (fr) 2020-12-21 2022-06-30 10X Genomics, Inc. Procédés, compositions et systèmes pour capturer des sondes et/ou des codes à barres
WO2022182682A1 (fr) 2021-02-23 2022-09-01 10X Genomics, Inc. Analyse à base de sonde d'acides nucléiques et de protéines
EP4301870A1 (fr) 2021-03-18 2024-01-10 10X Genomics, Inc. Capture multiplex de gène et expression de protéines à partir d'un échantillon biologique
WO2023034489A1 (fr) 2021-09-01 2023-03-09 10X Genomics, Inc. Procédés, compositions et kits pour bloquer une sonde de capture sur un réseau spatial
CN116635538A (zh) 2021-10-22 2023-08-22 塞弗德公司 诊断和治疗结核病的组合物和方法
US20230193310A1 (en) 2021-12-10 2023-06-22 Seminis Vegetabe Seeds, Inc. Lettuce plants having resistance to downy mildew
US20240035016A1 (en) 2022-04-29 2024-02-01 Cepheid Nucleic acid extraction and isolation with heat labile silanes and chemically modified solid supports
US20240017255A1 (en) 2022-05-19 2024-01-18 Cepheid Mvp cartridge and methods of use and manufacture
US20230404003A1 (en) 2022-06-21 2023-12-21 Seminis Vegetable Seeds, Inc. Novel qtls conferring resistance to cucumber mosaic virus
WO2024059692A1 (fr) 2022-09-15 2024-03-21 Abbott Laboratories Méthodes et produits de diagnostic, de pronostic et de thérapie du vhb

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4988617A (en) * 1988-03-25 1991-01-29 California Institute Of Technology Method of detecting a nucleotide change in nucleic acids
WO1993017126A1 (fr) * 1992-02-19 1993-09-02 The Public Health Research Institute Of The City Of New York, Inc. Nouvelles configurations d'oligonucleotides et utilisation de ces configurations pour le tri, l'isolement, le sequençage et la manipulation des acides nucleiques
US5407798A (en) * 1990-10-16 1995-04-18 Ciba Corning Diagnostics Corp. Amplification of midivariant DNA templates
US5536649A (en) * 1993-05-11 1996-07-16 Becton, Dickinson And Company Decontamination of nucleic acid amplification reactions using uracil-N-glycosylase (UDG)
US5593840A (en) * 1993-01-27 1997-01-14 Oncor, Inc. Amplification of nucleic acid sequences

Family Cites Families (124)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5258506A (en) * 1984-10-16 1993-11-02 Chiron Corporation Photolabile reagents for incorporation into oligonucleotide chains
US4883750A (en) * 1984-12-13 1989-11-28 Applied Biosystems, Inc. Detection of specific sequences in nucleic acids
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4925785A (en) * 1986-03-07 1990-05-15 Biotechnica Diagnostics, Inc. Nucleic acid hybridization assays
US5418149A (en) * 1990-07-24 1995-05-23 Hoffmann-La Roche Inc. Reduction of non-specific amplification glycosylase using DUTP and DNA uracil
US5202231A (en) * 1987-04-01 1993-04-13 Drmanac Radoje T Method of sequencing of genomes by hybridization of oligonucleotide probes
US5525464A (en) 1987-04-01 1996-06-11 Hyseq, Inc. Method of sequencing by hybridization of oligonucleotide probes
WO1989006700A1 (fr) 1988-01-21 1989-07-27 Genentech, Inc. Amplification et detection de sequences d'acides nucleiques
GB8810400D0 (en) * 1988-05-03 1988-06-08 Southern E Analysing polynucleotide sequences
US5700637A (en) 1988-05-03 1997-12-23 Isis Innovation Limited Apparatus and method for analyzing polynucleotide sequences and method of generating oligonucleotide arrays
US5185243A (en) * 1988-08-25 1993-02-09 Syntex (U.S.A.) Inc. Method for detection of specific nucleic acid sequences
AU629845B2 (en) 1988-08-30 1992-10-15 Abbott Laboratories Detection and amplification of target nucleic acid sequences
AU636875B2 (en) 1989-03-17 1993-05-13 Abbott Laboratories Method and device for improved reaction kinetics in nucleic acid hybridizations
AU5358990A (en) 1989-03-21 1990-10-22 Collaborative Research Inc. Multiplex dna diagnostic test
US5035996A (en) * 1989-06-01 1991-07-30 Life Technologies, Inc. Process for controlling contamination of nucleic acid amplification reactions
US5527681A (en) 1989-06-07 1996-06-18 Affymax Technologies N.V. Immobilized molecular synthesis of systematically substituted compounds
US5424186A (en) * 1989-06-07 1995-06-13 Affymax Technologies N.V. Very large scale immobilized polymer synthesis
US5744101A (en) 1989-06-07 1998-04-28 Affymax Technologies N.V. Photolabile nucleoside protecting groups
US6040138A (en) 1995-09-15 2000-03-21 Affymetrix, Inc. Expression monitoring by hybridization to high density oligonucleotide arrays
US5143854A (en) 1989-06-07 1992-09-01 Affymax Technologies N.V. Large scale photolithographic solid phase synthesis of polypeptides and receptor binding screening thereof
GB8920097D0 (en) * 1989-09-06 1989-10-18 Ici Plc Amplification processes
US5104792A (en) * 1989-12-21 1992-04-14 The United States Of America As Represented By The Department Of Health And Human Services Method for amplifying unknown nucleic acid sequences
US5516663A (en) 1990-01-26 1996-05-14 Abbott Laboratories Ligase chain reaction with endonuclease IV correction and contamination control
JPH04222599A (ja) 1990-04-20 1992-08-12 Syntex Usa Inc 二重受容体ポリヌクレオチド検定方法
JP3889807B2 (ja) 1990-05-03 2007-03-07 コーネル・リサーチ・ファンデーション・インコーポレイテッド 遺伝病を検出するための熱安定リガーゼによるｄｎａ増幅系
US5494810A (en) 1990-05-03 1996-02-27 Cornell Research Foundation, Inc. Thermostable ligase-mediated DNA amplifications system for the detection of genetic disease
US5484699A (en) * 1990-09-28 1996-01-16 Abbott Laboratories Nucleotide sequences useful as type specific probes, PCR primers and LCR probes for the amplification and detection of human papilloma virus, and related kits and methods
US5700636A (en) * 1990-10-19 1997-12-23 Becton Dickinson And Company Methods for selectively detecting microorganisms associated with vaginal infections in complex biological samples
CA2055755A1 (fr) 1990-11-22 1992-05-23 Toshihiko Kishimoto Methode d'immobilisation de l'adn a un seul brin sur un porteur au terminal
EP0834576B1 (fr) * 1990-12-06 2002-01-16 Affymetrix, Inc. (a Delaware Corporation) Détection de séquences d'acides nucléiques
DE4039348A1 (de) 1990-12-10 1992-06-11 Henkel Kgaa Teppichreinigungsmittel
WO1992010566A1 (fr) 1990-12-13 1992-06-25 Board Of Regents, The University Of Texas System Sondes d'hybridation in situ servant a l'identification et au marcage par bandes des chromosomes et regions chromosomiques specifiques chez l'homme
EP0566670A4 (en) * 1990-12-17 1993-12-08 Idexx Laboratories, Inc. Nucleic acid sequence detection by triple helix formation
US5290925A (en) * 1990-12-20 1994-03-01 Abbott Laboratories Methods, kits, and reactive supports for 3' labeling of oligonucleotides
RU1794088C (ru) * 1991-03-18 1993-02-07 Институт Молекулярной Биологии Ан@ Ссср Способ определени нуклеотидной последовательности ДНК и устройство дл его осуществлени
WO1992021079A1 (fr) * 1991-05-24 1992-11-26 The President And Fellows Of Harvard College Reacteur sequentiel et parallele
US5278298A (en) * 1991-05-29 1994-01-11 Merck & Co., Inc. Eimeria brunetti 16s rDNA probes
GB9112251D0 (en) 1991-06-07 1991-07-24 Amersham Int Plc Quantitative detection of nucleic acid amplification products
EP0519338B1 (fr) * 1991-06-20 1996-08-28 F. Hoffmann-La Roche Ag Méthodes améliorées d'amplification d'acides nucléiques
US5371241A (en) * 1991-07-19 1994-12-06 Pharmacia P-L Biochemicals Inc. Fluorescein labelled phosphoramidites
WO1993004199A2 (fr) 1991-08-20 1993-03-04 Scientific Generics Limited Procedes de detection et de quantification d'acides nucleiques, et de production d'acides nucleiques marques, immobilises
WO1993008472A1 (fr) 1991-10-15 1993-04-29 Multilyte Limited Methode de dosage par liaison a l'aide d'un reactif marque
CA2122450C (fr) 1991-11-01 2004-07-13 Charles Phillip Morris Processus d'amplification en phase solide
US5594121A (en) 1991-11-07 1997-01-14 Gilead Sciences, Inc. Enhanced triple-helix and double-helix formation with oligomers containing modified purines
US5412087A (en) 1992-04-24 1995-05-02 Affymax Technologies N.V. Spatially-addressable immobilization of oligonucleotides and other biological polymers on surfaces
US5324633A (en) * 1991-11-22 1994-06-28 Affymax Technologies N.V. Method and apparatus for measuring binding affinity
EP0633944B1 (fr) 1992-03-31 2000-11-08 Abbott Laboratories Procede de reaction de ligase en chaine multiplex
US5470705A (en) * 1992-04-03 1995-11-28 Applied Biosystems, Inc. Probe composition containing a binding domain and polymer chain and methods of use
ATE173767T1 (de) * 1992-04-03 1998-12-15 Perkin Elmer Corp Proben zusammensetzung und verfahren
AU4387193A (en) 1992-05-29 1993-12-30 Abbott Laboratories Ligase chain reaction starting with rna sequences
CA2115342C (fr) 1992-06-17 2003-08-26 Robert B. Wallace Methode de detection et de separation de sequences d'acide nucleique
US5981176A (en) 1992-06-17 1999-11-09 City Of Hope Method of detecting and discriminating between nucleic acid sequences
WO1994001446A2 (fr) 1992-07-09 1994-01-20 Beckman Instruments, Inc. Support solide organique derive utilise pour effectuer la synthese d'acides nucleiques
GB9214873D0 (en) 1992-07-13 1992-08-26 Medical Res Council Process for categorising nucleotide sequence populations
EP0654093B1 (fr) 1992-08-03 2001-01-03 Abbott Laboratories Detection et amplification d'acides nucleiques cibles par activite exonucleolytique
US6180338B1 (en) 1992-08-04 2001-01-30 Beckman Coulter, Inc. Method, reagent and kit for the detection and amplification of nucleic acid sequences
WO1994006906A1 (fr) 1992-09-18 1994-03-31 Merck & Co., Inc. ADN CODANT L'ENZYME DE CONVERSION DE L'INTERLEUKINE 1β PRECURSEUR MURIN
WO1994008047A1 (fr) 1992-09-25 1994-04-14 Abbott Laboratories Procede d'amplification en chaine par ligase pour la detection de petites mutations
WO1994009022A1 (fr) * 1992-10-09 1994-04-28 Oncor, Inc. PROCEDE DE DETECTION D'ANOMALIES CHROMOSOMIQUES STRUCTURALES PAR HYBRIDATION in situ DES TISSUS FIXES
US5795714A (en) * 1992-11-06 1998-08-18 Trustees Of Boston University Method for replicating an array of nucleic acid probes
JP2575270B2 (ja) * 1992-11-10 1997-01-22 浜松ホトニクス株式会社 核酸の塩基配列決定方法、単一分子検出方法、その装置及び試料の作成方法
US5605798A (en) * 1993-01-07 1997-02-25 Sequenom, Inc. DNA diagnostic based on mass spectrometry
WO1994016105A1 (fr) 1993-01-15 1994-07-21 The Public Health Research Institute Of The City Of New York, Inc. Dosages d'arn au moyen de sondes binaires d'arn et d'une ribozyme-ligase
EP0688366B1 (fr) 1993-01-15 2002-05-22 The Public Health Research Institute Of The City Of New York, Inc. Titrages sensibles par hybridation en sandwich de l'acide nucleique et kits afferents
AU6027794A (en) 1993-01-15 1994-08-15 Public Health Research Institute Of The City Of New York, Inc., The Diagnostic assays and kits for rna using rna binary probes and an rna-directed rna ligase
WO1994017206A1 (fr) * 1993-01-27 1994-08-04 Oncor, Inc. Procede d'amplification de sequences d'acides nucleiques
AU6092394A (en) * 1993-01-27 1994-08-15 Oncor, Inc. Amplification of nucleic acid sequences
US5985548A (en) 1993-02-04 1999-11-16 E. I. Du Pont De Nemours And Company Amplification of assay reporters by nucleic acid replication
DE69413574T2 (de) * 1993-02-09 1999-05-12 Agfa Gevaert Nv Eine Verarbeitungslösung und Verfahren zur Herstellung einer lithographischen Offsetdruckplatte nach dem Silbersalz-Diffusionsübertragungsverfahren
US5593826A (en) 1993-03-22 1997-01-14 Perkin-Elmer Corporation, Applied Biosystems, Inc. Enzymatic ligation of 3'amino-substituted oligonucleotides
US5422252A (en) * 1993-06-04 1995-06-06 Becton, Dickinson And Company Simultaneous amplification of multiple targets
US6709813B1 (en) 1993-05-14 2004-03-23 Ortho-Clinical Diagnostics, Inc. Diagnostic compositions, elements, methods and test kits for amplification and detection of human CMV DNA using primers having matched melting temperatures
US5652106A (en) * 1993-06-04 1997-07-29 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Rapid amplification-based subtyping of mycobacterium tuberculosis
DK72493D0 (da) 1993-06-18 1993-06-18 Risoe Forskningscenter Solid supports for use in peptide synthesis and assays
US5858659A (en) 1995-11-29 1999-01-12 Affymetrix, Inc. Polymorphism detection
US5837832A (en) 1993-06-25 1998-11-17 Affymetrix, Inc. Arrays of nucleic acid probes on biological chips
US5731171A (en) * 1993-07-23 1998-03-24 Arch Development Corp. Sequence independent amplification of DNA
US5601978A (en) 1993-09-03 1997-02-11 Abbott Laboratories Oligonucleotides and methods for the detection of chlamydia trachomatis
US5415839A (en) 1993-10-21 1995-05-16 Abbott Laboratories Apparatus and method for amplifying and detecting target nucleic acids
US6156501A (en) 1993-10-26 2000-12-05 Affymetrix, Inc. Arrays of modified nucleic acid probes and methods of use
US5352582A (en) * 1993-10-28 1994-10-04 Hewlett-Packard Company Holographic based bio-assay
US5429807A (en) 1993-10-28 1995-07-04 Beckman Instruments, Inc. Method and apparatus for creating biopolymer arrays on a solid support surface
US5415087A (en) * 1994-01-21 1995-05-16 Norfolk Southern Railway Co. Mobile tie banding apparatus
CA2190090A1 (fr) 1994-05-13 1995-11-23 Jon D. Kratochvil Materiaux et procedes de detection de microbacteries
US5631130A (en) * 1994-05-13 1997-05-20 Abbott Laboratories Materials and methods for the detection of Mycobacterium tuberculosis
US5508168A (en) * 1994-05-16 1996-04-16 Hoffmann-La Roche Inc. Methods and reagents for the detection of herpes simplex virus, treponema pallidum, and haemophilus ducreyi
US7378236B1 (en) 1994-06-17 2008-05-27 The Board Of Trustees Of The Leland Stanford Junior University Method for analyzing gene expression patterns
US5876924A (en) * 1994-06-22 1999-03-02 Mount Sinai School Of Medicine Nucleic acid amplification method hybridization signal amplification method (HSAM)
US5942391A (en) * 1994-06-22 1999-08-24 Mount Sinai School Of Medicine Nucleic acid amplification method: ramification-extension amplification method (RAM)
EP0717782A1 (fr) * 1994-06-22 1996-06-26 Mount Sinai School Of Medicine Of The City University Of New York Amplification dependant de la liaison s'appliquant a la detection d'agents pathogenes infectieux et de genes anormaux
US5834181A (en) 1994-07-28 1998-11-10 Genzyme Corporation High throughput screening method for sequences or genetic alterations in nucleic acids
DE69528706T2 (de) * 1994-08-19 2003-06-12 Pe Corp Ny Foster City Gekoppeltes ampflikation- und ligationverfahren
US5648213A (en) 1994-08-30 1997-07-15 Beckman Instruments, Inc. Compositions and methods for use in detection of analytes
US5695934A (en) * 1994-10-13 1997-12-09 Lynx Therapeutics, Inc. Massively parallel sequencing of sorted polynucleotides
US5604097A (en) * 1994-10-13 1997-02-18 Spectragen, Inc. Methods for sorting polynucleotides using oligonucleotide tags
US5512441A (en) * 1994-11-15 1996-04-30 American Health Foundation Quantative method for early detection of mutant alleles and diagnostic kits for carrying out the method
WO1996015271A1 (fr) 1994-11-16 1996-05-23 Abbott Laboratories Amplification multiplex dependant des ligatures
DE69637354T2 (de) 1995-06-05 2008-11-27 Biomerieux, Inc. Vorrichtung und verfahren zum detektieren von mikroorganismen
US5667974A (en) 1995-06-07 1997-09-16 Abbott Laboratories Method for detecting nucleic acid sequences using competitive amplification
EP0832287B1 (fr) 1995-06-07 2007-10-10 Solexa, Inc Marqueurs oligonuclotidiques servant a trier et a identifier
US5728526A (en) 1995-06-07 1998-03-17 Oncor, Inc. Method for analyzing a nucleotide sequence
US5723320A (en) 1995-08-29 1998-03-03 Dehlinger; Peter J. Position-addressable polynucleotide arrays
US5759779A (en) 1995-08-29 1998-06-02 Dehlinger; Peter J. Polynucleotide-array assay and methods
US5695937A (en) * 1995-09-12 1997-12-09 The Johns Hopkins University School Of Medicine Method for serial analysis of gene expression
US5856096A (en) 1995-09-20 1999-01-05 Ctrc Research Foundation Rapid and sensitive assays for detecting and distinguishing between processive and non-processive telomerase activities
US5854033A (en) 1995-11-21 1998-12-29 Yale University Rolling circle replication reporter systems
DE69612013T2 (de) 1995-11-21 2001-08-02 Univ Yale New Haven Unimolekulare segmentamplifikation und bestimmung
EP0880598A4 (fr) 1996-01-23 2005-02-23 Affymetrix Inc Evaluation rapide de difference d'abondance d'acides nucleiques, avec un systeme d'oligonucleotides haute densite
US6962263B2 (en) * 1996-01-24 2005-11-08 Sambrailo Packaging, Inc. Produce packaging system having produce containers with double-arched ventilation channels
US20020150921A1 (en) * 1996-02-09 2002-10-17 Francis Barany Detection of nucleic acid sequence differences using the ligase detection reaction with addressable arrays
US6852487B1 (en) * 1996-02-09 2005-02-08 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using the ligase detection reaction with addressable arrays
EP1958955B1 (fr) 1996-02-09 2013-09-04 Cornell Research Foundation, Inc. Réseaux d'oligonucleotides adressables spatialement et méthode pour leur fabrication
US5868136A (en) 1996-02-20 1999-02-09 Axelgaard Manufacturing Co. Ltd. Medical electrode
EP0912761A4 (fr) * 1996-05-29 2004-06-09 Cornell Res Foundation Inc Detection de differences dans des sequences d'acides nucleiques utilisant une combinaison de la detection par ligase et de reactions d'amplification en chaine par polymerase
US6312892B1 (en) 1996-07-19 2001-11-06 Cornell Research Foundation, Inc. High fidelity detection of nucleic acid differences by ligase detection reaction
WO1998036763A1 (fr) 1997-02-25 1998-08-27 Eli Lilly And Company Traitement de l'infertilite au moyen de ligands des recepteurs de leptine
US6313048B1 (en) * 1997-03-03 2001-11-06 Micron Technology, Inc. Dilute cleaning composition and method for using same
US5932711A (en) 1997-03-05 1999-08-03 Mosaic Technologies, Inc. Nucleic acid-containing polymerizable complex
US6899889B1 (en) * 1998-11-06 2005-05-31 Neomend, Inc. Biocompatible material composition adaptable to diverse therapeutic indications
US6506594B1 (en) 1999-03-19 2003-01-14 Cornell Res Foundation Inc Detection of nucleic acid sequence differences using the ligase detection reaction with addressable arrays
US7014994B1 (en) * 1999-03-19 2006-03-21 Cornell Research Foundation,Inc. Coupled polymerase chain reaction-restriction-endonuclease digestion-ligase detection reaction process
EP1165839A2 (fr) 1999-03-26 2002-01-02 Whitehead Institute For Biomedical Research Reseaux universels
US20030215821A1 (en) 1999-04-20 2003-11-20 Kevin Gunderson Detection of nucleic acid reactions on bead arrays
US6858412B2 (en) 2000-10-24 2005-02-22 The Board Of Trustees Of The Leland Stanford Junior University Direct multiplex characterization of genomic DNA

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4988617A (en) * 1988-03-25 1991-01-29 California Institute Of Technology Method of detecting a nucleotide change in nucleic acids
US5407798A (en) * 1990-10-16 1995-04-18 Ciba Corning Diagnostics Corp. Amplification of midivariant DNA templates
WO1993017126A1 (fr) * 1992-02-19 1993-09-02 The Public Health Research Institute Of The City Of New York, Inc. Nouvelles configurations d'oligonucleotides et utilisation de ces configurations pour le tri, l'isolement, le sequençage et la manipulation des acides nucleiques
US5593840A (en) * 1993-01-27 1997-01-14 Oncor, Inc. Amplification of nucleic acid sequences
US5536649A (en) * 1993-05-11 1996-07-16 Becton, Dickinson And Company Decontamination of nucleic acid amplification reactions using uracil-N-glycosylase (UDG)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP0912761A4 *

Cited By (161)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6458530B1 (en) 1996-04-04 2002-10-01 Affymetrix Inc. Selecting tag nucleic acids
US9400259B2 (en) 1996-04-25 2016-07-26 Bioarray Solutions, Ltd. Method of making a microbead array with attached biomolecules
US8691594B2 (en) 1996-04-25 2014-04-08 Bioarray Solutions, Ltd. Method of making a microbead array with attached biomolecules
EP0956359A1 (fr) * 1996-07-19 1999-11-17 Cornell Research Foundation, Inc. Detection tres precise de differences d'acides nucleiques par reaction de detection par ligase
EP0956359A4 (fr) * 1996-07-19 2004-12-01 Cornell Res Foundation Inc Detection tres precise de differences d'acides nucleiques par reaction de detection par ligase
US7244831B2 (en) 1996-07-19 2007-07-17 Cornell Research Foundation, Inc. High fidelity detection of nucleic acid differences by ligase detection reaction
US9902951B2 (en) 1997-04-01 2018-02-27 Illumina, Inc. Method of nucleic acid amplification
US8993271B2 (en) 1997-04-01 2015-03-31 Illumina, Inc. Method of nucleic acid amplification
US9593328B2 (en) 1997-04-01 2017-03-14 Illumina, Inc. Method of nucleic acid amplification
US6607878B2 (en) 1997-10-06 2003-08-19 Stratagene Collections of uniquely tagged molecules
US9399795B2 (en) 1998-06-24 2016-07-26 Illumina, Inc. Multiplex decoding of array sensors with microspheres
US8541218B2 (en) 1998-10-30 2013-09-24 Cornell Research Foundation, Inc. High fidelity thermostable ligase and uses thereof
US6949370B1 (en) 1998-10-30 2005-09-27 Cornell Research Foundation, Inc. High fidelity thermostable ligase and uses thereof
US9127269B2 (en) 1998-10-30 2015-09-08 Cornell Research Foundation, Inc. High fidelity thermostable ligase and uses thereof
US6395486B1 (en) 1999-03-15 2002-05-28 Applera Corporation Probe/mobility modifier complexes for multiplexnucleic acid detection
US6734296B1 (en) 1999-03-15 2004-05-11 Applera Corporation Probe/mobility modifier complexes for multiplex nucleic acid detection
WO2000055368A3 (fr) * 1999-03-15 2001-04-05 Perkin Elmer Corp Complexes sonde/modificateur de mobilite pour detection d'acide nucleique multiplex
WO2000055368A2 (fr) * 1999-03-15 2000-09-21 Pe Corporation (Ny) Complexes sonde/modificateur de mobilite pour detection d'acide nucleique multiplex
JP2003510011A (ja) * 1999-03-19 2003-03-18 コーネル リサーチ ファンデーション インク． カップル性ポリメラーゼ連鎖反応−制限エンドヌクレアーゼ消化−リガーゼ検出反応法
US9441267B2 (en) 1999-04-20 2016-09-13 Illumina, Inc. Detection of nucleic acid reactions on bead arrays
US9279148B2 (en) 1999-04-20 2016-03-08 Illumina, Inc. Detection of nucleic acid reactions on bead arrays
US6964847B1 (en) 1999-07-14 2005-11-15 Packard Biosciences Company Derivative nucleic acids and uses thereof
EP1194592A4 (fr) * 1999-07-14 2004-12-08 Packard Bioscience Company Acides nucleiques modifies et utilisations associees
EP1194592A1 (fr) * 1999-07-14 2002-04-10 Packard Bioscience Company Acides nucleiques modifies et utilisations associees
US7550276B2 (en) 1999-10-04 2009-06-23 Olympus Corporation Method of detecting nucleic acid
EP1136568A1 (fr) * 1999-10-04 2001-09-26 Olympus Optical Corporation Limited Procede de detection d'acide nucleique
EP1136568A4 (fr) * 1999-10-04 2004-12-15 Olympus Optical Corp Ltd Procede de detection d'acide nucleique
EP1221491A1 (fr) * 1999-10-12 2002-07-10 Precision System Science Co., Ltd. Systeme de suspension pour le sequen age d'une substance genetique, procede de sequen age d'une substance genetique mettant en oeuvre ledit systeme de suspension et procede d'identification des polymorphismes nucleotidiques uniques (snp) au moyen dudit systeme de suspension
EP1221491A4 (fr) * 1999-10-12 2003-04-16 Prec System Science Co Ltd Systeme de suspension pour le sequen age d'une substance genetique, procede de sequen age d'une substance genetique mettant en oeuvre ledit systeme de suspension et procede d'identification des polymorphismes nucleotidiques uniques (snp) au moyen dudit systeme de suspension
US8906626B2 (en) 2000-02-07 2014-12-09 Illumina, Inc. Multiplex nucleic acid reactions
US10837059B2 (en) 2000-02-07 2020-11-17 Illumina, Inc. Multiplex nucleic acid reactions
WO2001057268A2 (fr) * 2000-02-07 2001-08-09 Illumina, Inc. Detection d'acides nucleiques et procedes utilisant l'amorçage universel
JP2003521252A (ja) * 2000-02-07 2003-07-15 イルミナ インコーポレイテッド ユニバーサルプライミングを用いる核酸検出方法
WO2001057269A2 (fr) * 2000-02-07 2001-08-09 Illumina, Inc. Procedes de detection d'acide nucleique par amorçage universel
US6812005B2 (en) 2000-02-07 2004-11-02 The Regents Of The University Of California Nucleic acid detection methods using universal priming
US9850536B2 (en) 2000-02-07 2017-12-26 Illumina, Inc. Multiplex nucleic acid reactions
EP1990428A1 (fr) * 2000-02-07 2008-11-12 Illumina, Inc. Procédés de détection d'acide nucléique utilisant un amorçage universel
WO2001057268A3 (fr) * 2000-02-07 2002-07-18 Illumina Inc Detection d'acides nucleiques et procedes utilisant l'amorçage universel
WO2001057269A3 (fr) * 2000-02-07 2002-07-18 Illumina Inc Procedes de detection d'acide nucleique par amorçage universel
US6890741B2 (en) 2000-02-07 2005-05-10 Illumina, Inc. Multiplexed detection of analytes
US7361488B2 (en) 2000-02-07 2008-04-22 Illumina, Inc. Nucleic acid detection methods using universal priming
EP1130113A1 (fr) * 2000-02-15 2001-09-05 Johannes Petrus Schouten Méthode d'amplification dépendant de ligatures multiples
US6955901B2 (en) * 2000-02-15 2005-10-18 De Luwe Hoek Octrooien B.V. Multiplex ligatable probe amplification
WO2001061033A3 (fr) * 2000-02-15 2002-03-28 Johannes Petrus Schouten Amplification de sonde multiplex pouvant etre ligaturee
US7803537B2 (en) 2000-02-16 2010-09-28 Illumina, Inc. Parallel genotyping of multiple patient samples
US7285384B2 (en) 2000-02-16 2007-10-23 Illuminia, Inc. Parallel genotyping of multiple patient samples
US7482443B2 (en) * 2000-03-09 2009-01-27 Genetag Technology, Inc. Systems and methods to quantify and amplify both signaling probes for cDNA chips and genes expression microarrays
US7455965B2 (en) 2000-04-14 2008-11-25 Cornell Research Foundation, Inc. Method of designing addressable array for detection of nucleic acid sequence differences using ligase detection reaction
US10131938B2 (en) 2000-04-14 2018-11-20 Cornell Research Foundation, Inc. Method of designing addressable array suitable for detection of nucleic acid sequence differences using ligase detection reaction
US9340834B2 (en) 2000-04-14 2016-05-17 Cornell Research Foundation, Inc. Method of designing addressable array suitable for detection of nucleic acid sequence differences using ligase detection reaction
US9725759B2 (en) 2000-04-14 2017-08-08 Cornell Research Foundation, Inc. Method of designing addressable array suitable for detection of nucleic acid sequence differences using ligase detection reaction
US8492085B2 (en) 2000-04-14 2013-07-23 Cornell Research Foundation, Inc. Method of designing addressable array suitable for detection of nucleic acid sequence differences using ligase detection reaction
WO2001085987A1 (fr) * 2000-05-09 2001-11-15 Diatech Pty. Ltd. Methodes d'identification des zones repetitives de polynucleotidiques d'une longueur determinee
WO2001092579A2 (fr) * 2000-05-30 2001-12-06 Pe Corporation (Ny) Methodes de detection d'acides nucleiques cibles au moyen d'une ligation et d'une amplification couplees
WO2001092579A3 (fr) * 2000-05-30 2003-03-20 Pe Corp Ny Methodes de detection d'acides nucleiques cibles au moyen d'une ligation et d'une amplification couplees
US9709559B2 (en) 2000-06-21 2017-07-18 Bioarray Solutions, Ltd. Multianalyte molecular analysis using application-specific random particle arrays
WO2002002815A1 (fr) * 2000-07-05 2002-01-10 Toyo Boseki Kabushiki Kaisha Procede de detection de polymorphismes nucleotidiques
EP1229128A1 (fr) * 2001-01-31 2002-08-07 Boehringer Mannheim Gmbh Nouveau procédé pour la détermination du génotype
WO2002061122A2 (fr) * 2001-01-31 2002-08-08 Roche Diagnostics Gmbh Nouvelle methode de determination du genotype
WO2002061122A3 (fr) * 2001-01-31 2002-11-14 Roche Diagnostics Gmbh Nouvelle methode de determination du genotype
EP1247815A3 (fr) * 2001-03-25 2003-01-29 Exiqon A/S Oligonucléotides modifiés et leurs utilisations
EP1247815A2 (fr) * 2001-03-25 2002-10-09 Exiqon A/S Oligonucléotides modifiés et leurs utilisations
US9436088B2 (en) 2001-06-21 2016-09-06 Bioarray Solutions, Ltd. Un-supported polymeric film with embedded microbeads
US10415081B2 (en) 2001-10-15 2019-09-17 Bioarray Solutions Ltd. Multiplexed analysis of polymorphic loci by concurrent interrogation and enzyme-mediated detection
US7510829B2 (en) 2001-11-19 2009-03-31 Affymetrix, Inc. Multiplex PCR
EP1456416A2 (fr) * 2001-11-19 2004-09-15 Parallele Bioscience, Inc. Amplification multiplex cible par attache d'oligonucleotides
US10407717B2 (en) 2001-11-19 2019-09-10 Affymetrix, Inc. Methods of analysis of methylation
US7754451B2 (en) 2001-11-19 2010-07-13 Affymetrix, Inc. Multiplex oligonucleotide addition and target amplification
EP1456416A4 (fr) * 2001-11-19 2006-01-11 Parallele Bioscience Inc Amplification multiplex cible par attache d'oligonucleotides
US10822642B2 (en) 2001-11-19 2020-11-03 Affymetrix, Inc. Methods of analysis of methylation
US7208295B2 (en) 2001-11-19 2007-04-24 Affymetrix, Inc. Multiplex oligonucleotide addition and target amplification
EP1319718A1 (fr) * 2001-12-14 2003-06-18 Keygene N.V. Analyse et détection de séquences cibles multiples à haut rendement
WO2003052140A2 (fr) * 2001-12-14 2003-06-26 Keygene N.V. Analyse a rendement eleve et detection de sequences cibles multiples
WO2003052140A3 (fr) * 2001-12-14 2003-11-13 Keygene Nv Analyse a rendement eleve et detection de sequences cibles multiples
WO2003060163A2 (fr) * 2001-12-28 2003-07-24 Keygene N.V. Discrimination et detection de sequences nucleotidiques cibles utilisant la spectrometrie de masse
WO2003060163A3 (fr) * 2001-12-28 2003-12-11 Keygene Nv Discrimination et detection de sequences nucleotidiques cibles utilisant la spectrometrie de masse
EP2210665A1 (fr) 2002-01-16 2010-07-28 CLONDIAG GmbH Appareil de détection des molecules ciblées
US9251583B2 (en) 2002-11-15 2016-02-02 Bioarray Solutions, Ltd. Analysis, secure access to, and transmission of array images
US8592214B2 (en) 2003-01-21 2013-11-26 Illumina, Inc. Chemical reaction monitor
US7887752B2 (en) 2003-01-21 2011-02-15 Illumina, Inc. Chemical reaction monitor
EP2266699A1 (fr) 2003-04-02 2010-12-29 CLONDIAG GmbH Appareil pour l'amplification et la détection d'acide nucléique
US7799525B2 (en) 2003-06-17 2010-09-21 Human Genetic Signatures Pty Ltd. Methods for genome amplification
US8808991B2 (en) 2003-09-02 2014-08-19 Keygene N.V. Ola-based methods for the detection of target nucleic avid sequences
WO2005021794A2 (fr) * 2003-09-02 2005-03-10 Keygene N.V. Procedes bases sur l'amplification ou le dosage d'une ligation d'oligonucleotide (ola) permettant de detecter des sequences d'acide nucleique cibles
WO2005021794A3 (fr) * 2003-09-02 2005-05-19 Keygene Nv Procedes bases sur l'amplification ou le dosage d'une ligation d'oligonucleotide (ola) permettant de detecter des sequences d'acide nucleique cibles
EP1668148A1 (fr) * 2003-09-04 2006-06-14 Human Genetic Signatures PTY Ltd. Dosage de detection d'acide nucleique
US7846693B2 (en) 2003-09-04 2010-12-07 Human Genetic Signatures Pty. Ltd. Nucleic acid detection assay
EP1668148A4 (fr) * 2003-09-04 2007-11-14 Human Genetic Signatures Pty Dosage de detection d'acide nucleique
US8691754B2 (en) 2003-09-22 2014-04-08 Bioarray Solutions, Ltd. Microparticles with enhanced covalent binding capacity and their uses
US9637777B2 (en) 2003-10-28 2017-05-02 Bioarray Solutions, Ltd. Optimization of gene expression analysis using immobilized capture probes
EP2241639A2 (fr) 2004-01-26 2010-10-20 CLONDIAG GmbH Procédé de génotypage et de pathotyage de Pseudomonas aeruginosa
EP1718766A2 (fr) * 2004-02-10 2006-11-08 Cornell Research Foundation, Inc. Procede de detection d'etat de methylation de promoteur
US7358048B2 (en) 2004-02-10 2008-04-15 Cornell Research Foundation, Inc. Method for detection of promoter methylation status
EP1718766A4 (fr) * 2004-02-10 2009-05-27 Cornell Res Foundation Inc Procede de detection d'etat de methylation de promoteur
WO2005092038A3 (fr) * 2004-03-22 2006-06-01 Univ Johns Hopkins Procedes de detection de differences d'acides nucleiques
WO2005092038A2 (fr) * 2004-03-22 2005-10-06 The Johns Hopkins University Procedes de detection de differences d'acides nucleiques
US8679788B2 (en) 2004-03-22 2014-03-25 The Johns Hopkins University Methods for the detection of nucleic acid differences
US8168777B2 (en) 2004-04-29 2012-05-01 Human Genetic Signatures Pty. Ltd. Bisulphite reagent treatment of nucleic acid
WO2005118862A3 (fr) * 2004-04-30 2006-07-13 Applera Corp Compositions, techniques et kits permettant d'effectuer ou de mal effectuer une ligature d'oligoleotides
WO2005118862A2 (fr) * 2004-04-30 2005-12-15 Applera Corporation Compositions, techniques et kits permettant d'effectuer ou de mal effectuer une ligature d'oligoleotides
EP2299257A2 (fr) 2004-05-06 2011-03-23 CLONDIAG GmbH Dispositif et procédé pour détecter des interactions moléculaires
EP3527287A1 (fr) 2004-05-06 2019-08-21 Alere Technologies GmbH Procéde pour de détecter des interactions moléculaires
EP3171155A1 (fr) 2004-05-06 2017-05-24 Clondiag GmbH Dispositif et procede pour de detecter des interactions moleculaires
EP2290351A2 (fr) 2004-05-06 2011-03-02 CLONDIAG GmbH Dispositif et procédé pour détecter des interactions moléculaires
EP2280267A2 (fr) 2004-05-06 2011-02-02 CLONDIAG GmbH Dispositif et procédé pour de detecter des interactions moleculaires
EP2256478A2 (fr) 2004-05-06 2010-12-01 CLONDIAG GmbH Dispositif et procede pour de detecter des interactions moleculaires
WO2006005055A2 (fr) * 2004-06-30 2006-01-12 Applera Corporation Procedes, melanges reactionnels et trousses destinees a la ligature de polynucleotides
WO2006005055A3 (fr) * 2004-06-30 2006-03-16 Applera Corp Procedes, melanges reactionnels et trousses destinees a la ligature de polynucleotides
US9147037B2 (en) 2004-08-02 2015-09-29 Bioarray Solutions, Ltd. Automated analysis of multiplexed probe-target interaction patterns: pattern matching and allele identification
US7803580B2 (en) 2004-09-10 2010-09-28 Human Genetic Signatures Pty. Ltd. Amplification blocker comprising intercalating nucleic acids (INA) containing intercalating pseudonucleotides (IPN)
US7833942B2 (en) 2004-12-03 2010-11-16 Human Genetic Signatures Pty. Ltd. Methods for simplifying microbial nucleic acids by chemical modification of cytosines
US8598088B2 (en) 2004-12-03 2013-12-03 Human Genetic Signatures Pty. Ltd. Methods for simplifying microbial nucleic acids by chemical modification of cytosines
US8431347B2 (en) 2005-05-26 2013-04-30 Human Genetic Signatures Pty Ltd Isothermal strand displacement amplification using primers containing a non-regular base
US8343738B2 (en) 2005-09-14 2013-01-01 Human Genetic Signatures Pty. Ltd. Assay for screening for potential cervical cancer
EP2330215A1 (fr) 2005-11-04 2011-06-08 CLONDIAG GmbH Procédé et dispositif de détection d'interactions moléculaires
US8236498B2 (en) 2006-01-20 2012-08-07 Olympus Corporation Method of detecting nucleotide sequence with an intramolecular probe
WO2008118998A2 (fr) * 2007-03-27 2008-10-02 Primera Biosystems Inc. Procédé de détection multiplex et de quantification d'acides nucléiques
WO2008118998A3 (fr) * 2007-03-27 2008-11-27 Primera Biosystems Inc Procédé de détection multiplex et de quantification d'acides nucléiques
US8685675B2 (en) 2007-11-27 2014-04-01 Human Genetic Signatures Pty. Ltd. Enzymes for amplification and copying bisulphite modified nucleic acids
EP2356245A4 (fr) * 2008-11-07 2013-02-20 Univ Utah Res Found Biais d'amplification d'allèleallele amplification bias
US9422597B2 (en) 2008-11-07 2016-08-23 Biofire Diagnostics, Inc. Allele amplification bias
US10351903B2 (en) 2008-11-07 2019-07-16 University Of Utah Research Foundation Allele amplification bias
EP2356245A1 (fr) * 2008-11-07 2011-08-17 University Of Utah Research Foundation Biais d'amplification d'allèleallele amplification bias
US10422001B2 (en) 2011-07-08 2019-09-24 Keygene N.V. Sequence based genotyping based on oligonucleotide ligation assays
WO2013009175A1 (fr) 2011-07-08 2013-01-17 Keygene N.V. Génotypage à base de séquence en fonction d'analyses de ligature d'oligonucléotides
US11873529B2 (en) 2011-07-08 2024-01-16 Keygene N.V. Sequence based genotyping based on oligonucleotide ligation assays
US10988807B2 (en) 2011-07-08 2021-04-27 Keygene N.V. Sequence based genotyping based on oligonucleotide ligation assays
EP2980226A1 (fr) 2011-07-08 2016-02-03 Keygene N.V. Génotypage à base de séquence en fonction d'analyses de ligature d'oligonucléotides
US9777322B2 (en) 2011-07-08 2017-10-03 Keygene N.V. Sequence based genotyping based on oligonucleotide ligation assays
US9732375B2 (en) 2011-09-07 2017-08-15 Human Genetic Signatures Pty. Ltd. Molecular detection assay using direct treatment with a bisulphite reagent
WO2013095119A1 (fr) 2011-12-14 2013-06-27 De Staat Der Nederlanden, Vert. Door De Minister Van Vws Identification de souches de poliovirus
EP3287531A1 (fr) * 2012-02-28 2018-02-28 Agilent Technologies, Inc. Procédé de fixation d'une séquence de compteur pour un échantillon d'acide nucléique
WO2013128281A1 (fr) * 2012-02-28 2013-09-06 Population Genetics Technologies Ltd Procédé de fixation d'une contre-séquence à un échantillon d'acides nucléiques
US9670529B2 (en) 2012-02-28 2017-06-06 Population Genetics Technologies Ltd. Method for attaching a counter sequence to a nucleic acid sample
US10626441B2 (en) 2012-02-28 2020-04-21 Agilent Technologies, Inc. Method for attaching a counter sequence to a nucleic acid sample
US9551038B2 (en) 2012-05-11 2017-01-24 K-Mac System for integrated analysis of real-time polymerase chain reaction and DNA chip and method for integrated analysis using the same
CN103388023A (zh) * 2012-05-11 2013-11-13 科美仪器 实时聚合酶链反应的集成分析系统和dna芯片以及使用其的集成分析方法
CN103388023B (zh) * 2012-05-11 2015-09-16 科美仪器 实时聚合酶链反应的集成分析系统和dna芯片以及使用其的集成分析方法
US10059983B2 (en) 2012-09-10 2018-08-28 Genesky Diagnostics (Suzhou) Inc. Multiplex nucleic acid analysis
EP2893034A4 (fr) * 2012-09-10 2016-04-13 Genesky Diagnostics Suzhou Inc Procédé d'analyse d'acide nucléique multiplexe
EP2893034A1 (fr) * 2012-09-10 2015-07-15 Genesky Diagnostics (Suzhou) Inc. Procédé d'analyse d'acide nucléique multiplexe
WO2015185564A1 (fr) * 2014-06-02 2015-12-10 Base4 Innovation Ltd Procédé de détection de polymorphismes nucléotidiques
CN106460057A (zh) * 2014-06-02 2017-02-22 贝斯4创新公司 核苷酸多态性检测方法
US10227638B2 (en) 2014-06-02 2019-03-12 Base4 Innovation Ltd. Nucleotide polymorphism detection method
AU2015330844B2 (en) * 2014-10-08 2022-02-03 Cornell University Method for identification and quantification of nucleic acid expression, splice variant, translocation, copy number, or methylation changes
US11466311B2 (en) 2014-10-08 2022-10-11 Cornell University Method for identification and quantification of nucleic acid expression, splice variant, translocation, copy number, or methylation changes
CN107002145A (zh) * 2014-10-08 2017-08-01 康奈尔大学 用于使用针对遗留预防的组合核酸酶、连接反应和聚合酶反应鉴定和相对定量核酸序列表达、剪接变体、易位、拷贝数或甲基化变化的方法
EP4026914A1 (fr) * 2014-10-08 2022-07-13 Cornell University Procédé d'identification et de quantification relative de changements de méthylation de séquence d'acide nucléique au moyen de réactions combinées de nucléase, de ligature et de polymérase comportant une prévention de transfert
EP3204520A4 (fr) * 2014-10-08 2018-03-07 Cornell University Procédé pour l'identification et la détermination quantitative de l'expression d'acide nucléique, variant d'épissage, translocation, nombre de copies ou modifications de méthylation
EP3690060A1 (fr) * 2014-10-08 2020-08-05 Cornell University Procédé d'identification et de quantification relative d'expression de séquence d'acide nucléique, de variant d'épissage, de translocation, de nombre de copies ou de changements de méthylation en utilisant des réactions combinées de nucléase, de ligature et de polymérase comportant une prévention de transfert
US10344321B2 (en) 2014-10-08 2019-07-09 Cornell University Method for identification and quantification of nucleic acid expression, splice variant, translocation, copy number, or methylation changes
US11542506B2 (en) 2014-11-14 2023-01-03 Voyager Therapeutics, Inc. Compositions and methods of treating amyotrophic lateral sclerosis (ALS)
JP2016140288A (ja) * 2015-01-30 2016-08-08 倉敷紡績株式会社 一本鎖ｄｎａ産物の調製方法
WO2016121907A1 (fr) * 2015-01-30 2016-08-04 倉敷紡績株式会社 Procédé de préparation d'un produit d'adn monocaténaire
WO2018127408A1 (fr) 2017-01-05 2018-07-12 Tervisetehnoloogiate Arenduskeskus As Quantification de séquences d'adn
WO2018172348A1 (fr) * 2017-03-21 2018-09-27 Sequencing Multiplex Sl Amplification et marquage faciles en une seule étape (eosal)
EP3378950A1 (fr) * 2017-03-21 2018-09-26 Sequencing Multiplex SLK Amplification et marquage simples en une étape
US11603542B2 (en) 2017-05-05 2023-03-14 Voyager Therapeutics, Inc. Compositions and methods of treating amyotrophic lateral sclerosis (ALS)
US11434502B2 (en) 2017-10-16 2022-09-06 Voyager Therapeutics, Inc. Treatment of amyotrophic lateral sclerosis (ALS)
WO2019238765A1 (fr) 2018-06-12 2019-12-19 Keygene N.V. Procédé d'amplification d'acide nucléique
WO2020169830A1 (fr) 2019-02-21 2020-08-27 Keygene N.V. Génotypage de polyploïdes

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